Determination of Tobramycin using HPAE

Determination of tobramycin using HPAE-IPAD
on a compact ion chromatography system
Terri Christison and Jeff Rohrer
Thermo Fisher Scientific,
Sunnyvale, CA
To develop an HPAE-IPAD method for the determination of kanamycin A,
kanamycin B, and tobramycin using a high-pressure-capable
Thermo Scientific™ Dionex™ Integrion™ HPIC™ system.
Integrion, CarboPac PA1,
aminoglycoside, carbohydrate,
antibiotic, drug substance
Tobramycin is an important aminoglycoside antibiotic used in ophthalmic
and intravenous treatments to treat bacterial infections by blocking protein
synthesis.1 Tobramycin is isolated from the fermentation of the actinomycete
Streptomyces tenebrarius with kanamycin A and kanamycin B present as
impurities from either incomplete isolation of the drug or from degradation of
tobramycin. Therefore, it is important to assay the tobramycin content and
quantify the related impurities of a tobramycin-based antibiotic. Determination
of tobramycin, kanamycin, and other aminoglycoside antibiotics has
been previously demonstrated by High Performance Anion Exchange
with Integrated Pulsed Amperometric Detection (HPAE-IPAD).2–6 These
publications demonstrated the advantages of using eluent generation (EG) for
this application.
This document updates Thermo Scientific™ Application Note 61 using a highpressure-capable Dionex Integrion HPIC system equipped with updated EG
technology and an electrochemical detector.
•Thermo Scientific Dionex Integrion HPIC System
(P/N 22153-60305), including:
––Temperature control in the detector and column oven
––EG capabilities
––Eluent degas module
––Device monitoring
––Vacuum Degas Kit (P/N 00108-01-00046)
•Integrion ED detector (P/N 22153-62035) with
Thermo Scientific™ Dionex™ Integrion electrochemical
cell without electrodes (P/N 072044) (or the same P/N
from the Thermo Scientific™ Dionex™ ICS-3000 IC,
ICS-5000 IC or ICS-5000+ HPIC systems)
•Mobile Control Option (P/N 22153-62031) and Android
tablet (P/N 22153-62100)
•Thermo Scientific™ Dionex™ AS-AP Autosampler
temperature control option (P/N 074926) with
1.5 mL trays (P/N 074936) and 250 µL syringe
(P/N 074306)
•Degassed ASTM™ Type I deionized water,7 vacuum
degassed with ultrasonic agitation
•pH buffer solutions to calibrate pH – Ag/AgCl Reference
electrode: Fisher Scientific™ P/N SB115-500 (pH 7) and
P/N SB107-500 (pH 10)
•Tobramycin (Sigma-Aldrich® Chemical Co,
P/N T40014)
•Kanamycin B (Sigma-Aldrich Chemical Co,
P/N B5264)
Thermo Scientific™ Chromeleon™ Chromatography Data
System (CDS) software, version 7.2 SR4
Table 1 lists the consumable products needed for a
Dionex Integrion HPIC Reagent-Free IC capabilities,
configured for electrochemical detection.
Chromatographic Conditions
Thermo Scientific™ Dionex™
CarboPac™ PA1 guard (4 × 50 mm)
and separation (4 × 250 mm)
2 mM KOH
Eluent Source: Thermo Scientific™ Dionex™
EGC 500™ KOH cartridge with
Thermo Scientific™ Dionex™
CR-ATC™ 600 trap column and
high pressure EG degas module
(P/N 075522)
Flow Rate:
0.5 mL/min
30 °C
30 °C
Injection Volume:20 µL, in PushFull mode
Time (s) Potential (V) Integration
0.21+0.33 Begin
0.56+0.33 End
IPAD, AAA-Direct Waveform vs pH,
1.67 Hz
AAA-Direct disposable gold
working electrode, 0.002” thick
Teflon® gasket
pH/Ag/AgCl in pH mode
Run Time: 16 min
Background: 50–90 nC
Table 1. Consumables list.
Thermo Scientific™ Dionex™
IC PEEK Viper™ fitting tubing
assembly kits
Dionex IC PEEK Viper fitting assembly kit for the Dionex
Integrion HPIC system: Includes one each of
P/Ns: 088805–088807, 088809, 088811
Guard to separator column: 0.007 × 4.0 in (102 mm)
Injection Valve, Port C (Port 2) to guard column:
0.007 × 5.5 in long (140 mm)
EGC Eluent Out to CR-TC Eluent In: 0.007 × 6.5 in (165 mm)
Separator to ED Cell In: 0.007 × 7.0 in (178 mm)
CR-TC Eluent Out to Degasser Eluent In: 0.007 × 9.5 in
(241 mm)
Dionex AS-AP Autosampler vials
Package of 100, polystyrene vials, caps, blue septa,10 mL
Dionex AS-AP Autosampler vials
Package of 100, polypropylene vials, caps, 1.5 mL
Thermo Scientific™ Dionex™
EGC™ 500 KOH Eluent Generator
Eluent generator cartridge
Recommended for carbohydrate analysis methods using eluent
EG Vacuum Degas Conversion Kit generation. Included when ordering a Dionex Integrion HPIC
system with ED detector
Dionex IC PEEK Viper fitting
tubing assemblies
Part Number
HP EG Degas module
Recommended for use with eluent generation. Included when
ordering an Integrion with eluent generation
Thermo Scientific™ Dionex™
CR-ATC™ 600 Electrolytic
trap column
Continuously regenerated trap column used on Integrion
systems with the Dionex EGC KOH 500 cartridge
Thermo Scientific™ Dionex™
CarboPac™ PA1 Guard
Guard column, 4 × 50 mm
Dionex CarboPac PA1
separation column
Separation column, 4 × 250 mm
Thermo Scientific™ Dionex™
AAA-Direct disposable working
AAA-Direct carbohydrate gold working electrodes, preferred for
this application
Thermo Scientific™ Dionex™
Carbohydrate disposable working
Carbohydrate gold working electrodes, suggested alternative
working electrodes for this application
Thermo Scientific™ Dionex™
Ag/AgCl reference electrode
Reference electrode for this application
Extra gaskets for working
Extra gaskets, PTFE, 0.002” thick
Thermo Scientific™ Nalgene™
Rapid Flow™ sterile disposable
filter units
1000 mL vacuum filtration flask, PES membrane, 0.2 µm,
(Fisher Scientific
P/N 09-741-03)
Standard and eluent preparation
Use ASTM Type I deionized (DI) water for standards,
eluent, and autosampler flush solution. It is important to
degas the DI water to supply the eluent generator and to
maintain 3–5 psi of inert gas headspace over the DI water
eluent when running an HPAE-PAD method. Absorbed
carbon dioxide gas can result in poor chromatography
and variable retention times.
To prepare 2 L of the degassed ASTM Type I DI water
to supply the eluent generator, degas 1 L of DI water by
vacuum filtration (Nalgene, 1 L, PES, 0.2 µm) with applied
ultrasonic agitation. Transfer the degassed water to the
2 L eluent bottle and cap the bottle. Prepare another
1 L in the same manner. Connect the 2 L eluent bottle
containing degassed DI water to the Dionex Integrion
HPIC system pump eluent line. Connect nitrogen or
other inert gas to the eluent bottle to provide ~3–5 psi
headspace pressure.
Weigh the tobramycin reagent and dissolve in DI water to
prepare a 10 mg/mL stock standard. Use polypropylene
volumetric flasks, storage containers, and autosampler
vials to avoid sample losses. Tobramycin, and to a lesser
extent kanamycin B, when dissolved in water adsorbs
Fresh ASTM Type I
Fresh Deionized Water
to glass surfaces. Significant losses due to adsorption
occur at dilute concentrations.2 The tobramycin working
standards were prepared by diluting the stock standard
with DI water to 0.5, 1.0, 10, 50, and 100 µM. Kanamycin
A and kanamycin B solutions (0.1, 1, 5, and 10 µM) were
prepared in the same manner. For recovery experiments,
10 µM and 26 µM tobramycin solutions were prepared
from the stock standards. Method detection limit (MDL)
standards were prepared by serial dilution from the
0.1 µM working standard. The samples were stored at
-20 °C and thawed prior to analysis.
Instrument setup and installation
The Dionex Integrion HPIC System with RFIC capabilities
is a high-pressure-capable, integrated system. The
Dionex Integrion HPIC system, the Dionex EGC 500 KOH
cartridge and Dionex CR-ATC 600 consumable products
are designed for high pressure conditions up to 5000 psi.
To set up this application, connect the Dionex
AS-AP autosampler and the Dionex Integrion HPIC
system, equipped with an electrochemical detector,
according to Figure 1. Note that the injection valve is
plumbed through different ports than previous Dionex IC
systems. See Thermo Scientific TN 176.8
Non-Metallic Pump
Dionex EGC 500
CR-ATC 600
Deionized Water
Data Management
Guard and Separation Columns
Dionex AS-AP
(sample injection)
Sample Loop
Figure 1. Flow diagram for the Dionex Integrion HPIC System configured for ED detection.
Connect the USB cables from the Dionex Integrion HPIC
system to the Dionex AS-AP autosampler and to the
computer. Connect the power cables and power on the
IC instrument and the autosampler.
Configuring the modules in the Chromeleon CDS
To configure the IC system:
1.Start the Chromeleon Instrument Controller program
and select the link, Configure Instruments (opens
Chromeleon Instrument Configuration Manager).
2.Right-click on computer name.
3.Select Add an Instrument, and enter an appropriate
name (for example: Integrion_Tobramycin_1).
4.Select Add a Module, IC: Dionex Integrated Modules,
and Integrion HPIC System.
In this application, only three modules are needed: the
Dionex Integrion IC system, the autosampler, and the
Integrion Pump Wellness module. The instructions to
configure each module are summarized in Table 2.
To add the Dionex Integrion IC system in the
1.Right-click and select Add a Module, IC: Dionex
Integrated Modules, Integrion HPIC System module.
2.Select USB address to link the module to the
The Chromeleon CDS software will automatically
detect all Dionex Integrion IC system devices—the
electrolytic devices, detectors, pump degasser, and seal
wash—requiring minimal data entry during instrument
configuration. The Chromeleon CDS software automates
the system configuration process by automatically
detecting these installed devices (Figure 2).
Table 2. Summary of system configuration for the Dionex Integrion HPIC system.
Dionex Integrion HPIC Module
Link to USB address
Flow Rate and pressure limitations are displayed
Automatically detected
Automatically detects Dionex eluent generator cartridges, and Dionex CR-TC trap columns
(Figure 2)
Inject Device
Automatically detected
Thermal Controls
Automatically detects thermal control options for column, and detector
High-Pressure Valves
Automatically detected
Low-Pressure Valves
Not needed for this application, but automatically detected.
Automatically detects Pump Degasser and Seal Wash pump
Pump Wellness Module
Click pressure signal box
Activates pressure monitoring feature (Figure 3)
Dionex AS-AP Autosampler Module
Add module
Link to USB address
Only if more than one instrument is detected. If this option is present, select Instrument
Segments / Pump Link
Select 10 mL polystyrene vials or 1.5 mL vials for “Red”, “Blue”, and “Green”
Select Push, select syringe size, select 1.2 mL buffer line, enter the loop size
Adding the Dionex AS-AP Autosampler to the
1.Add the Dionex AS-AP Autosampler as a module, and
select the USB address.
2.In the Segments/Pump Link tab, select the appropriate
vial trays for each color zone.
3.In the Options tab, select Push, installed syringe size,
1.2 mL for buffer line, and enter the sample loop
volume (20 µL).
4.Save the configuration.
5.Select Check the Configuration.
Figure 2. Automatic detection of electrolytic devices in the Dionex
Integrion HPIC System module.
To add pressure monitoring capabilities:
1. Right-click and select Add a Module, IC: Dionex
Integrated Modules, Integrion HPIC Pump Wellness
2. Select USB address to link the module to the
3. Select the Devices tab and click on the pressure signal
box (Figure 3).
6.Close the Chromeleon Instrument Configuration
Plumbing the Dionex Integrion HPIC System
Decontaminate the IC system prior to installing the
columns by puping a 500 mM NaOH solution at
0.5 mL/min from the pump to valves for 2–3 h, or
overnight if the system was previously used for another
application. Finish the decontamination process by
pumping DI water at 0.5 mL/min for 30 min.
To achieve the best chromatography, use the Dionex IC
PEEK Viper fitting assemblies (listed below) and tighten to
finger-tight. It is important that they are not overtightened.
These fittings are used in the following locations:
•Dionex EGC 500 KOH eluent generator cartridge Eluent Out to Eluent In on Dionex CR-ATC 600 trap
•Dionex CR-ATC 600 trap column - Eluent Out to Eluent
In on the Dionex Degas Module
•Injection Valve - Port 2 (Column) to the guard column
•Between the guard and separation columns
•Separation column to Eluent In on the ED
electrochemical cell
Figure 3. Adding the Dionex Integrion HPIC Pump Wellness
module to instrument configuration.
To install an HPAE-IPAD application on the Dionex
Integrion HPIC system, follow the instructions in Technical
Note 176.8
The degasser pump should turn on for a short time
and then shut off, indicating that the degasser pump is
working and that the vacuum is acceptable.
1.Loosen the waste lines, including the metal-wrapped
waste line in the back of the instrument, and direct the
free ends to a waste container.
To assist in degassing the eluent, install a ¼” i.d. air
tubing from the EG Degas module (P/N 075522) vent
port to the vacuum connection in the back of the Dionex
Integrion HPIC system (Figure 4). The degasser pump will
turn on again for a short time to re-establish the vacuum.
If the degasser pump stays on for longer than 1 min,
tighten the fitting (Figure 4) ¼ turn.
2.To plumb the system, first connect the pump eluent line
to the eluent bottle containing
DI water previously degassed (vacuum filtration and
ultrasonic agitation).
3.Apply 3 to 5 psi of nitrogen or other inert gas
headspace to prevent carbon dioxide absorption.
4.Prime the pump by opening the priming knob
¼ turn and pressing the priming button.
5.Prime the pump until no bubbles are visible and water
is flowing at a steady rate out of the pump waste line.
6.Turn off the pump priming and close the priming knob
to finger-tight.
For more information, review the Dionex Integrion HPIC
system Installation and Operator’s manual.9
Install vacuum line to degasser module vent
Hydroxide eluents produced by eluent generation
require inline vacuum degassing to achieve the optimum
conditions for electrochemical detection. Vacuum
degassing is accomplished by connecting the vent line of
the degas module to the vacuum pump port on the back
of the instrument (Figure 4). In the Dionex Integrion HPIC
system configured with an electrochemical detector (ED),
the vacuum pump connector is pre-installed, eliminating
the manual installation previously required.
At the start of the application, to ensure that the degasser
pump is working properly, reset the pump degasser to
“Off” and then “On”.
1.Press the “F8” key while on the instrument console
page, which brings up the manual commands.
Figure 4. Installing the vacuum connection on the Dionex
Integrion HPIC system.
2.Place the cursor on the pump and set Degasser to
Conditioning electrolytic devices and columns
3.Close the dialogue window and repeat with “On”.
Do not remove the tags on the columns and consumable
devices. These tags are required for consumable device
monitoring functionality.
Figure 5. Consumables online installation instructions.
Install the Dionex EGC 500 KOH cartridge and Dionex
CR-ATC 600 Continuously Regenerated Anion Trap
Column in the Reservoir Tray compartment. Condition the
devices according to instructions in the drop-down menu
under Consumables, Install (Figure 5). (This information
is also available in the product manuals and the system
installation manual.9-13)
3.Connect the autosampler syringe line to wash container
containing degassed DI water to the syringe.
Condition the columns for 30 min according to the
instructions from the Consumables, Install Column
section (Figure 5). The general practice is follow the
eluent and flow rate conditions listed in the column’s
Quality Assurance Report (QAR) while directing the eluent
exiting the column to a waste container. Complete the
installation according to the flow diagram in Figure 1.
6.Calibrate the transfer line volume by following the
prompts on the TLV Calibration button. This volume will
be recorded automatically.
Installing and optimizing the Dionex AS-AP
The Dionex AS-AP Autosampler needle must be aligned
to the injection port. To align the autosampler needle:
1.Select the Sampler tab on the instrument panel, and
press the Alignment button.
2.Follow the commands to align the autosampler needle
to the Injection Port and Wash Port (Section B.12 in the
Operator’s Manual).12
4.Prime the syringe to flush out any air in the Buffer Wash
line and syringe.
5.Initially select a 5000 μL wash volume until a steady
flow of water is observed at the Wash Port. Reset wash
volume to 100 μL.
For more information review Section 5.9 in the Dionex IC
Series AS-AP Autosampler Operator’s Manual.12
Electrochemical cell
Always wear particle-free nitrile gloves (such as Fisher
Scientific P/N 19-130-1597 series) when handling the
electrochemical cell. If this is a new ED Cell, disassemble
the cell and discard the shipping gasket. Caution: Do not
touch the working electrode with any paper products,
as this can contaminate the working electrode. Avoid
wrinkles in the gasket.
The ED Cell is a three-electrode cell: the cell body as the
counter electrode, a reference electrode (pH-Ag/AgCl),
and a working electrode (conventional or disposable). The
fully assembled cell also includes a Yoke Block Assembly
(with a support block) to tighten the cell and gaskets
for the working electrode. The installation procedures
mentioned below describe an electrochemical cell with
an AAA-Direct gold disposable working electrode.
Installing the disposable electrode
1.Rinse the cell body, the walls of the reference
electrode, and the inlet tube thoroughly with DI water.
2.Shake off the excess water, and dry with a lab wipe.
3.Select the working electrode for the application and
corresponding gasket and support block. Rinse them
with DI water and shake off the excess water.
4.Dry the gasket (0.002” thick Teflon) and support block
with an absorbent tissue.
5.Assemble the cell according to the Operator’s manual7
and ED User’s Compendium for Electrochemical
Detection13 by first installing the working electrode
gasket flat against cell body. Avoid any wrinkles in the
gasket, as this will cause a poor fit and subsequent
leaks and poor detection. To minimize the chance of
using a worn gasket and developing a leak, the gasket
should be replaced after three compressions (i.e.
disposable electrode installations).
6.Install the AAA-Direct disposable working electrode
with the metal face down over the gasket.
7.Install the support block firmly over the working
8.Install the Yoke Block by squeezing the tabs and sliding
it on the cell body.
9.Align the Yoke Block parallel to the cell body and rotate
the Yoke Block knob clockwise until you hear three
“clicks”. (The cell with a conventional working electrode
is assembled similarly with appropriate gasket, except
the support block is not used.)
Calibrating and installing the pH-Ag/AgCl
reference electrode in the electrochemical cell
To calibrate the reference electrode:
1.Condition the pH-Ag/AgCl reference electrode by
removing the storage cap, rinsing the electrode with
DI water to remove the potassium chloride storage
solution, and then placing the electrode in a solution of
pH 7 buffer.
2.Select pH buffer 7 and the corresponding buffer for the
application, pH 10 for basic eluents and pH 4 for acidic
3.Install the cell into ED module and connect the yellow
cable to the yellow port.
4.Install the reference electrode blue cable into the black
5.Immerse the reference electrode in pH 7 buffer to at
least mid-level of the electrode.
6.Select the “pH Calibration” button on the ED Panel
and follow the instructions to calibrate the electrode
including using pH 10 buffer.
If calibration fails, it will be reported in the audit trail.
To install the reference electrode into the cell:
1.After calibration is completed and to make the
installation more convenient, first remove the reference
electrode and cell body from the ED module.
2.Rinse the buffer solution off the electrode with
DI water, and gently, but firmly, screw-in or rotate
the reference electrode clockwise into the reference
electrode port of the electrochemical cell until the
reference electrode is finger tight.
3.Immediately complete the final plumbing as described
Completing the plumbing
For optimum cell performance, use 2–3 ft (60–90 cm)
of black PEEK tubing or 1–2 in (5–10 cm) of red PEEK
tubing at 0.5 mL/min for optimum cell backpressure of
~12 psi at 0.5 mL/min.
Caution: Excess tubing can cause band-broadening and
thus reduce detection response.
The cell waste line should be removed prior to each
reference electrode installation and installed after flow
has started through the cell. (See discussion below.)
Complete the installation:
1.Install the cell in the ED detector module and connect
the reference electrode cable (blue to black) and the
counter/working electrode cable (yellow to yellow).
2.Remove the temporary waste line from the column
and install the IC PEEK Viper fitting (P/N 088809,
0.007 × 7.0 in) to the column outlet.
3.Allow liquid to flow from the end of the column.
4.Connect the free end to the cell inlet tube.
5.Allow the liquid to flow from cell exit hole and then
connect a 2 to 3 ft piece of black PEEK tubing (0.010
in, 0.254 mm i.d.).
6.Wait 60 s before tightening the connection. The
tubing provides ~12 psi backpressure (at 0.5 mL/min)
on the cell to prevent outgassing and detector noise.
(The comparable length of red PEEK (0.005 in,
0.013 mm i.d.) tubing is 1–2 in.)
This application may require up to 1500 psi delivered
by backpressure tubing.
4.Set the eluent concentration, column oven,
compartment oven, and cell temperatures as shown in
the Conditions section of the application.
5.Turn on the ED cell after the pH > 10.
6.Select Integrated Pulsed Amperometric for cell mode.
7.Select AAA-Direct waveform.
8.Enter the data collection frequency and the “pH” as
reference electrode mode into the ED Panel.
9.Allow the system to equilibrate for 30 min.
For optimum chromatography equilibrate until the total
background is stable, ~5 nC/min.
Creating an instrument sequence and instrument
Use the Thermo Scientific™ AppsLab™ Library of
Analytical Application database to download the
Chromeleon sequence.14
To download the sequence:
1. Open AppsLab database.
2. Search for the title of this application note or search by
using the key words, tobramycin and Integrion.
3. Select the AppsLab record.
7.Connect the cell waste tubing to the CR-ATC Regen In
4. Select the Downloads tab.
Starting the Dionex Integrion HPIC System
5. Select the eWorkflow to automatically generate the
Chromeleon sequence.
To start the system:
1.Turn on the pump.
2. Immediately turn on both the Dionex EGC 500 eluent
generator cartridge and the Dionex CR-ATC 600 trap
column when liquid is flowing through the device.
The system backpressure is dependent on the flow
rate and type of column but must be above 2000 psi to
support the EGC cartridges.
3 Install yellow PEEK backpressure tubing (yellow PEEK,
0.076 mm i.d., 0.003 in i.d.) between the HP EG Degas
module and the injection port (Port 1, Pump position).
Use Chromeleon Wizard to create a new method and a
new sequence.
To create a new instrument method:
1.Select Create, Instrument Method, and select
Instrument (such as Integrion_Tobramycin_1).
2.Enter the values from the Chromatographic Conditions
3.Save the instrument method.
To create a new sequence:
1.Select Create, Sequence, select the instrument, select
ED or ED_Total as the preferred channel.
Review and approval of the devices is required to
start the first sequence on the Dionex Integrion HPIC
system and after installing new consumable devices.
To access this approval:
1.Select Consumables and select Inventory (Figure 6).
The device monitoring shows the device history,
tracking: Part No., size, serial numbers, manufacture
lot, installed location (On Device), and Best if Use by
Date (Figure 6, top). Additionally, the device monitoring
will provide warnings if there is incompatibility in the
devices installed (Figure 6, bottom left).
2.To start the sequence, review the list of consumables
listed as inventory.
3.Correct any errors and approve.
2.Save the sequence.
4.Close the page (Figure 6, bottom right).
Device monitoring
5.Select the Instrument Queue tab.
A new feature of the Dionex Integrion HPIC system is
the device monitoring and tracking, which automatically
detects the electrolytic devices and the columns.
6.Conduct a Ready Check on the sequence.
7.Press Start.
Figure 6. Consumables tracking.
Results and discussion
Dionex CarboPac PA1 guard, 4 × 50 mm
Dionex CarboPac PA1, 4 × 250 mm
2 mM KOH
Eluent Source:
Dionex EGC-500 KOH cartridge,
with Dionex CR-ATC 600 trap column,
Dionex high pressure degasser
Flow Rate:
0.5 mL/min
Column Temperature: 30 °C
Detector Compart.: 30 °C
Injection Volume:
20 µL
IPAD, AAA-Direct Au disposable electrode,
0.002” thick gasket
Reference Electrode: pH/Ag/AgCl, pH mode
AAA-Direct, versus pH , 1.67 Hz
1. Void volume
2. System peak
3. Kanamycin A
4. Kanamycin B
5. Tobramycin
6. Oxygen dip
Tobramycin and other aminoglycoside determinations
were previously demonstrated in AN61 using HPAE-PAD
or IPAD with an IC system, autosampler, and EG device.
Since AN61 was published, there have been many
technology advances in IC instruments, consumables,
and detectors. This application update demonstrates
the same application executed on a Dionex Integrion
HPIC system with an ED detector and ED cell, the latest
technology in eluent generation consumables, and a
Dionex AS-AP Autosampler.
Tobramycin and the aminoglycoside impurities
were separated by an electrolytically generated eluent
(2 mM KOH). Electrolytic eluent generation provides
accurate eluent concentrations and precise retention
times. The aminoglycosides were separated at
0.5 mL/min on the Dionex CarboPac PA1 anionexchange column and detected by IPAD using the
AAA-Direct waveform.
Figure 7 shows the chromatogram of tobramycin with
kanamycin impurities. The run-time was extended two
minutes from the AN61 conditions to 16 min to ensure
that the oxygen dip (a dip in the baseline that is result
of having less dissolved oxygen in the sample than in
the eluent) did not appear near a peak of interest. The
oxygen dip (~31-min retention time) is due to oxygen
present in the samples and appears as a function of the
gas permeation volume of the column. Like some organic
impurities, eluting oxygen produces less background
than the eluent, so there is a dip in the baseline. The
retention times of the oxygen dip and other baseline
dips vary from column to column, and depend on the
flow rate, not the eluent strength. Eluting the baseline
dips just prior to the end of run, or timing their elution to
occur at the end of the following injection, prevents the
baseline dips from interfering with the peaks of interest.
The tobramycin peak exhibits some asymmetry and a
small baseline slope after it elutes, but does not interfere
with integration. At the low eluent conditions, carbonate
can accumulate on the column resulting in a reduction
of analyte retention time. A prudent practice is to run a
column wash in the morning at 2 mM KOH for 16 min and
Figure 7. 20 µM tobramycin with trace amounts of kanamycin
A and B.
65 mM KOH for another 44 min. It may be necessary to
add another column wash during the day, but it was not
found to be necessary during these experiments.
The peak response of the tobramycin was also
evaluated using the Four Potential Carbohydrate
waveform. The response is significantly lower than with
the AAA-Direct waveform, as previously reported in
AN61, but if desired, the carbohydrate waveform can be
used as demonstrated and discussed in AN61.
Method qualification
Sensitivity was estimated by calculating the limit of
detection (LOD) using nine replicate injections of a
0.010 µM tobramycin standard (S/N 24), diluted serially
from 1 µM tobramycin working standard. The estimated
LOD at 3 × S/N was 1.3 nM (0.026 pmol on column,
20 µL injection), which is comparable to previously
reported values.
The LODs for kanamycin A and kanamycin B were
determined similarly using 10-fold dilution of 0.1 µM
working standards. The results were 14 (0.29 pmol on
column) and 15 µM (0.30 pmol on column), respectively.
Area (nC - min)
Amount, µM
Amount, µM
Amount, µM
Area (nC - min)
The peak response to concentration (linearity) was
evaluated from 1–100 µM (0.5, 1.0, 10, 50, and 100 µM)
for tobramycin and 0.1–10 µM (0.1, 1, 5, and 10 µM) for
kanamycin A and kanamycin B. The responses were
linear for both kanamycin analytes with coefficients of
determination, r2 > 0.999). These results were similar to
those reported in AN61. In contrast to previous reports,
the best correlation was obtained for tobramycin by
applying a quadratic fit (Figures 8A, 8B, 8C).
Area (nC - min)
The tobramycin method was evaluated on the Dionex
Integrion HPIC system using the previously described
conditions. Additionally, general system suitability
practices were followed as stated in USP® General
Chapter <621> Chromatography.15 The method was
evaluated for reproducibility, linearity, accuracy, and
sensitivity. The short-term stability was evaluated by
determining the reproducibility of triplicate injections
of 20 µM tobramycin and two of the impurities, 10 µM
kanamycin A and 5 µM kanamycin B. The experiments
showed good reproducibility with RSDs of 1.68, 0.46,
0.14%, respectively, for tobramycin, kanamycin A, and
kanamycin B.
Figure 8. Calibration curves for (A) tobramycin, (B) kanamycin A,
and (C) kanamycin B.
The method accuracy was determined by the
average percent recovery of six replicate injections of
16.8 ± 0.24 µM tobramycin added to a 10.05 ± 0.10 µM
standard. The experiments averaged 98.6% recovery,
indicating that the method was accurate.
Determination of kanamycin A, kanamycin B, and
tobramycin were demonstrated on a Dionex CarboPac
PA1 anion exchange column by HPAE-IPAD. In this
update, previously published AN61 is demonstrated
on the Dionex Integrion HPIC system. Additionally,
detailed instructions were added to support successful
installations and operation.
2. Thermo Scientific Application Note 61: Determination of Tobramycin and Impurities
Using HPAE-PAD, Sunnyvale, CA, November 2004.
The method was evaluated for linearity, reproducibility,
and sensitivity which were found to be comparable
to the values previously reported in AN61. All of the
aminoglycoside antibiotic compounds evaluated had
higher responses using the AAA-Direct waveform (Gold,
AAA) than the Four Potential Carbohydrate waveform.
However, as reported in AN61, the Four Potential
Carbohydrate waveform (Gold, Carbo, Quad) is also
suitable when pmol sensitivity is not needed.
More information on this application, including
downloadable instrument methods, can be accessed
through the Thermo Scientific AppsLab Library of
Analytical Applications.14
1. Hanko, V.P.; Rohrer, J.S.; Liu, H.H.; Zheng, C.; Zhang, S.; Liu, X.; Tang, X. Identification
of tobramycin impurities for quality control process monitoring using highperformance anion-exchange chromatography with integrated pulsed amperometric
detection. J. Pharm. Biomed. Anal., 2008, 47(4–5), 828–833.
3. Thermo Scientific Application Note 66: Determination of Neomycin B and Impurities
Using HPAE-PAD. Sunnyvale, CA, July 2006.
4. Thermo Scientific Application Note 186: Analysis of Paromomycin by HPAE-IPAD.
Sunnyvale, CA, October 2007.
5. Thermo Scientific Application Update 167: Determination of Tobramycin in Crude and
In-Process Production Samples During Manufacturing Using HPAE-IPAD, Sunnyvale,
CA, June 2009.
6. Hanko, V. P., Rohrer, J. S. Ion Chromatography Applications for Aminoglycoside
Antibiotics. In Applications of Ion Chromatography for Pharmaceutical and Biological
Products, Edition 1; Bhattacharyya, L., Rohrer, J. S.; John Wiley and Sons: New York,
Wiley Online Library, 2012; pp 175–192. DOI: 10.1002/9781118147009
7. ASTM International, ASTM D1193, Standard Specification of Water, West
Conshohocken, PA, 2006.
8. Thermo Scientific Technical Note 176: Configuring the Dionex Integrion HPIC System
for Fast Determinations of Monosaccharides and Disaccharides Using HPAE-PAD with
Eluent Generation. Sunnyvale, CA, 2016.
9. Thermo Fisher Scientific. Integrion Installation and Operator’s Manual.
P/N 22153-97003, Sunnyvale, CA, August 2016.
10.Thermo Fisher Scientific. Dionex Product Manual for Eluent Generator Cartridges.
P/N 065018-05, Sunnyvale, CA, June 2014.
11.Thermo Fisher Scientific. Dionex CarboPac PA1 Column Product manual.
P/N 065547-01, Sunnyvale, CA, November 2013.
12.Thermo Fisher Scientific. Dionex AS-AP Operator’s Manual. Document No. 065259,
Sunnyvale, CA, 2012.
13.Thermo Scientific Electrochemical Detection User’s Compendium, P/N: 065340-02,
April 2013.
14.Thermo Scientific AppsLab Library of Analytical Applications. [Online] https://appslab. (Accessed Dec. 9, 2016).
15. United States Pharmacopeia General Chapter <621> Chromatography, in USP
National Formulary (NF): USP 37, 2014.
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