Selectivity is the KEY
Mixed-mode chromatography addresses unmet challenges in pharmaceutical analysis:
API and counter ion by RP/AEX/CEX trimodal columns (e.g. Acclaim Trinity P1 and P2)
Unique Charged Aerosol Detector for consistent response of analytes that are weak or non-chromophoric molecules
Unmatched performance for counter ion analysis by dedicated column technology, unique charged aerosol detection and established UltiMate 3000 technology
New IonCount complete solution for ease of use and quick methods development of new API and counter ion analysis
Easy operation by predefined eWorkflows, Thermo Scientific™ Dionex™ Chromeleon™ 7.2 Chromatography Data System, and viper connection tubing
Chromatography: Pharmaceutical Analysis of API and Counter Ions in Complex Formulations in a Single Injection
1. 1
The world leader in serving science
Matthew Neely
Pittcon™ Conference & Expo 2014
March 2-6, 2014
Pharmaceutical Analysis of API and
Counter Ions in Complex Formulations
in a Single Injection
2. 2
Outline
• Challenges in pharmaceutical analysis
• Mixed-mode chromatography overview
• Mixed-mode chromatography for pharmaceutical analysis
• API and counter ions
• Thermo Scientific™ Dionex ™ Corona™ Veo™ Charged
Aerosol Detector
• Near universal, mass sensitive detector for routine LC determinations of
any non-volatile and many semi-volatile analytes
• Ion-Count Solutions
• Unmatched performance for counter ion analysis by dedicated column
technology, unique charged aerosol detection and established Thermo
Scientific™ Dionex™ Ultimate™ 3000 HPLC technology
• Summary
3. 3
Challenges in Pharmaceutical Analysis
• Pharmaceutical molecules are highly diverse: hydrophobicity, charge,
size, etc.
• Most drug candidates are highly polar; many have poor chromophores.
• Reversed-phase columns (e.g. C18) – the “work horse”
• Not suited for highly hydrophilic analytes
• Hydrophilic interaction (HILIC)/normal phase (NP) – the solution?
• Solubility challenge
• Sample matrix effect
• Method ruggedness
• Ion exchange chromatography – good for charged analytes
• Lack of retention for neutral analytes
• Lack of selectivity for analytes with same charge
• Possible solution…
4. 4
Mixed-Mode Chromatography
• Definition
• Hydrophobic interaction + ion-exchange interaction
• Benefits
• Adjustable selectivity
• Simplified mobile phase (no ion-pairing reagents)
• Simultaneous separation of different types of analytes
• Types
• Anion-exchange/reversed-phase (AEX/RP)
• Cation-exchange/reversed-phase (CEX/RP)
• Anion-exchange/cation-exchange/reversed-phase (AEX/CEX/RP)
5. 5
RP/IEX Bimodal Mixed-Mode Columns
Silica Gel Silica Gel Silica Gel
Ion-exchange Reversed-phase
Mixed beads Mixed ligands Single ligand
(embedded)
Single ligand
(tipped)
6. 6
RP/AEX/CEX Trimodal Mixed-Mode Columns
Cation-exchange
Anion-exchange
Mixed beads Single ligand
(amphoteric)
Nanopolymer Silica Hybrid
(NSH)
Silica Gel
CEX
RP/AEX
Silica GelSilica Gel
8. 8
Acclaim Trinity P1 vs. P2 Columns
Attributes Acclaim Trinity P1 Column Acclaim Trinity P2 Column
Column
chemistry
Nanopolymer Silica Hybrid Technology (NSH)
Retention
mechanism
RP/WAX/SCX HILIC/SAX/WCX
Counter ions High resolution for mono-valent
ions. Some capability for
multivalent ions
Most suitable for general screening
of a broad range of ions (both
mono- and multi-valents)
Simultaneous
determination of
API and counter
ions
Generally good for both
hydrophilic and hydrophobic APIs
and respective counter ions
Good for hydrophilic APIs and
counter ions
Other
applications
Generally applicable to any
applications that involve ionic
analytes, including both
hydrophobic and hydrophilic
ionics, and hydrophobic neutrals
Potential solution for any hydrophilic
molecules, neutral or ionic, such as
sugars
9. 9
Challenge: API and Counter Ions
• Salt formation is important in drug development
• 50% of all drugs are formulated as salt forms
• Challenges
• RP: little or no retention for counter ion; little to adequate retention
for API
• IEX: need both AEX and CEX columns
• HILIC: limited use
• Solution
• Acclaim Trinity P1 column– RP/AEX/CEX trimodal phase
• Acclaim Trinity P2 column – HILIC/WCX/SAX trimodal phase
• Designed for API and counter ion analysis
11. 11
Pharmaceutical-Related Anions and Cations –
Trinity P2 Column
Column: Acclaim Trinity P2, 3 µm
Dimensions: 3.0 x 50 mm
Mobile Phase: D.I. water and 100 mM NH4OFm, pH 3.65
Temperature: 30°C
Flow Rate: 0.60 mL/min
Inj. Volume: 1 µL
Detection: Corona Veo
Samples: 0.02 – 0.10 mg/mL each in D.I. water
Peaks:
1. Phosphate
2. Sodium
3. Potassium
4. Chloride
5. Malate
6. Bromide
7. Nitrate
8. Citrate
9. Fumarate
10. Sulfate
11. Magnesium
12. Calcium
Gradient:
Time
(min)
H2O 0.1 M
Ammonium
formate, pH3.65
-8 90 10
0 90 10
1 90 10
11 0 100
15 0 100
0 5 10 15
0.0
1.8
pA
Minutes
1
2
3
4
5
6
7
8
9
1011
12
12. 12
t0
0.6 mL/min
2.0 mL/min
Column: Acclaim Trinity P1, 3 µm
Dimensions: 3.0 x 50 mm
Mobile Phase: 80/20 v/v CH3CN/20 mM (total) NH4OAc, pH5
Temperature: 30 °C
Flow Rate: 0.6 and 2.0 mL/min
Inj. Volume: 2.5 µL
Detection: Corona ultra
(Gain = 100pA; Filter = med; Neb Temp = 30°C)
Sample: Na, Naproxen (0.2 mg/mL in mobile phase)
Peaks:
1. Na+
2. Naproxen
21
1
2
ONa
Minutes
0 2 431
mV
1000
t0
1
0
Naproxen Sodium – Trinity P1 Column
13. 13
API and Counter-Ions in Adderall – Trinity P2
Column
Column: Acclaim Trinity P2, 3 µm
Dimensions: 3.0 x 50 mm
LC System: UltiMate 3000 RSLC
Mobile Phases: A: Acetonitrile
B: Water
C: 100 mM Ammonium formate, pH 3.65
Gradient: -8.0 0.0 0.5 5.0 10.0 12.0
%A 35 35 35 35 20 20
%B 59 59 59 0 0 0
%C 6 6 6 65 80 80
Flow: 0.60 mL/min
Temperature: 30 °C
Injection: 5 µL
Detector: Diode array, UV 254 nm
Corona Veo (evaporator 55 °C, data rate 5 Hz,
filter 2 sec, power function 1.5)
Blank subtracted baseline.
Sample: Standards in 100 mM acetic acid; equivalent to
200 µg/mL Adderall-XR
Peaks: 1. Aspartate 24 µg/mL
2. Sodium
3. Saccharin 24
4. Amphetamine 122
5. Sulfate 26
0
20
mAU
UV
0 2 4 6 8 10 12
0
20
pA
Minutes
Charged aerosol
1
2
3
5
4
3
Adderall is indicated in ADHD treatment
14. 14
Why Use Mixed-Mode Columns?
• Retention
• Highly hydrophilic analytes, e.g. pharmaceutical counterions
• Selectivity
• Adjustable selectivity allows for easy method optimization
• Throughput
• Determining analytes of various charges and hydrophobicity with one
single injection
• MS-compatibility
• No need for ion-pairing reagent for hydrophilic charged analytes
• Method robustness
• More resistant to sample matrix effect than HILIC
15. 15
Corona Veo Detector—
The Next Generation Charged Aerosol Detector
• Uses the fastest growing and
most rapidly adopted universal LC
detection technology since diode
array detection (aka. PDA or DAD)
• Provides unbiased detection for a
wide variety of analyte classes
• New design incorporates unique,
proprietary technologies only
available from Thermo Fisher
Scientific
A novel, near universal, mass sensitive detector for routine
LC determinations of any non-volatile and many semi-volatile
analytes
16. 16
Introduction to Charged Aerosol Detection
Comparison of Charged Aerosol
Detection to UV and MS
• Used to quantitate any non-volatile and
many semi-volatile analytes with LC
• Provides consistent analyte response
independent of chemical structure and
molecule size
• Neither a chromophore, nor the ability to
ionize, is required for detection
• Dynamic range of over four orders of
magnitude from a single injection (sub-ng
to µg quantities on column)
• Mass sensitive detection – provides
relative quantification without the need
for reference standards
• Compatible with gradient conditions for
HPLC, UHPLC and Micro LC
17. 17
Particle Charging for Charged Aerosol Detection
Mixing Chamber
Dry analyte
particles
Charged
nitrogen gas
Analyte particles
with charged surface
• Charges remain on
surface of analyte
particles.
• Particles remain
intact and do not
ionize.
• The more surface
area, the more
charge is carried
by the particle
18. 18
Corona Veo Detector – What's New ?
• Radically new FocusJet™
concentric nebulization system
improves sensitivity and
precision
• All new evaporation scheme
widens the scope of
applications to include low flow
capabilities for capillary and
micro LC, as well as UHPLC
• Usability and serviceability are
enhanced by countless
improvements, many of which
came from our customers
This entirely new detector incorporates many design and
performance improvements:
19. 19
Corona Veo Detector with FocusJet Nebulizer
Technology
• The advanced concentric
nebulizer design ensures a
superior aerosol atomization
pattern for uniform droplet
formation
• Exchangeable design gives
users direct access for
improved serviceability and
ease-of-use
• Accepts typical inlet fittings,
including fingertight Thermo
Scientific™ Dionex™ Viper™
capillary fingertight fittings
FocusJet Concentric Nebulizer
Viper Fingertight Fitting
20. 20
FocusJet Concentric Nebulization System
• The inlet liquid and nebulization gas streams coaxially form into a stable
aerosol at the nebulizer tip
• Small droplets are transported upward into the heated evaporation sector
• Larger droplets fall and are expelled by a precision micro-pump
Coaxial N2 flow
Capillary Inlet
Aerosol
FocusJet Concentric Nebulizer Tip
Cross-flow Nebulizer Active drain pump
To Evaporation
Sector
Gas In (N2)Inlet
Concentric
Nebulizer
21. 21
Corona Veo SD/RS Detectors –
Product Differences
Model / Feature
Corona Veo
Detector
Corona Veo RS
Detector
Flow Rate Range 0.20 – 2.0 mL/min 0.01 – 2.0 mL/min
Evaporation Temp 35°C or 50°C Ambient +5°C to +100°C
Data Acquisition Rate Up to 100 Hz Up to 200 Hz with CM 7.x
Integrated Stream Switching
Valve (SSV)
-
6-port, 2-position micro
valve
Standalone Interface Integrated color LCD touch screen
Inlet Gas Pressure (Reqd) 4.8 - 5.5 bar (70-80 psig; 482 – 551 kPa)
Internal Gas Regulation Manual adjustment Electronic control
Liquid Waste Non-pressurized
Enhanced Linearity Via Power Function (User applied)
Analog Signal Output Optional Optional
22. 22
Electronic Gas Regulation –
Corona Veo RS Detector
• Provides ultra-precise gas pressure to ensure stable particle flow
• Ensures optimum detector performance over the entire inlet flow
range of the Corona Veo RS detector (0.01 – 2.0 mL/min)
• Analytical and Micro LC operation modes are selectable through
remote control via the chromatography data system
Internal Electronic Gas Regulator
23. 23
Integrated Stream Switching–
Corona Veo RS Detector
Stream Switching Valve (SSV)
• Externally accessible automated 6-port 2-position valve
• Diverts flow to waste in event of gas or pump flow errors
• Simplify system operations by enabling access to alternate devices
without the need to unplumb/replumb modules (UV, MS etc.)
• Improve chromatography results for complex samples by allowing
elimination of harmful salts or collection of unretained peaks
24. 24
• User Selectable Power Function Value (PFV)
• Increases the observed linear range of calibration data
• Acts to improve mass balance determinations
Power Function with Corona Veo Detectors
Extend the linear dynamic range using the Power Function
25. 25
Benefits of Charged Aerosol Detection
• Used to quantitate any non-volatile and many semi-volatile
analytes at sub-nanogram levels with LC
• Consistent analyte response is independent of chemical
structure and molecule size
• Neither a chromophore nor the ability to ionize is required for
detection
• Dynamic range of over four orders of magnitude within a
single injection (sub-ng to µg on column)
• Mass sensitive detection – provides relative quantification
without the need for reference standards
• Compatible with gradient conditions for HPLC, UHPLC and
Micro LC
26. 26
Corona Detector Summary
• Corona Veo Charged Aerosol detectors:
• Provides consistent inter-analyte responses across a wide
range
• Best frontline detector for weak and non-chromophore
analytes
• Ideal replacement for older, less capable universal
detection technologies (RI, ELSD, etc.)
• A complimentary detector to UV, Diode Array and MS
• Expands applications potential for future assay
development
• Improved linearity with power function
28. 28
Chromeleon 7.2: eWorkflows
1. Choose eWorkflow,
select instrument,
and click “Launch”
2. Enter number of
samples and start position
3. Sequence is created and can be
immediately run
29. 29
Predefined eWorflows for API and Counter Ion
• Purpose: API and counter ion analysis
• Generic organic gradient with isocratic ion strength (can be
fine tuned for specific problems)
• Resolving power for monovalent counter ions
0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 10,0
0,00
1,00
2,00
3,00
3,85
min
pA 1
2
3
4
5
6
Column: Acclaim Trinity P1, 3x50mm
System: UltiMate 3000 LPG SD
Mobile phase: A - H2O
B - ACN
C-200 mM NH4From, pH=3.65
Gradient: 0-14 min 5-85% B, 14-15 min 85% B,
15.0-15.5 min, 85-5% B, 15.5-22 min
5% B
0-22min 10% C
Flow rate: 700 L/min
Power Func.: 1.5
Temperature: 30 ºC
Injection: 1 µL
Detection: Corona Veo RS
Analytes: 1. Sodium
2. Potassium
3. Chloride
4. Bromide
5. Nitrate
6. Maleic Acid
30. 30
Predefined eWorflows for API and Counter Ion
• Purpose: API and counter ion analysis
• Generic organic gradient with isocratic ion strength (can be
fine tuned for specific problems)
• API (Cefazolin) with sodium as counter ion
Column: Acclaim Trinity P1, 3x50mm
System: UltiMate 3000 LPG SD
Mobile phase: A - H2O
B - ACN
C-200 mM NH4From, pH=3.65
Gradient: 0-14 min 5-85% B, 14-15 min 85% B,
15.0-15.5 min, 85-5% B, 15.5-22 min
5% B
0-22min 10% C
Flow rate: 700 L/min
Power Funct: 1.5
Temperature: 30 ºC
Injection: 1 µL
Detection: Corona Veo RS
Analytes: 1. Sodium
2. Cefazolin
0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 10,0
0,00
2,50
5,00
7,50
10,00
1
2
31. 31
Predefined eWorflows for Counter Ion Screening
• Purpose: Screening of a wide range of counter ions
• Broad ionic strength gradient (10 to 100 mM)
• Applicable to mono- and divalent ions
Column: Acclaim Trinity P2, 3x50mm
System: UltiMate 3000 LPG SD
Mobile phase: A - H2O
B - ACN
C-100 mM NH4From, pH=3.65
Gradient: 0-1 min 10% C, 1-11 min 10-100% C,
11-20 min 100% C, 20-21 100-10% C,
21-29 min 10% C
Flow rate: 600 L/min
Power Func.: 1.5
Temperature: 30 ºC
Injection: 1 µL
Detection: Corona Veo RS
Analytes: 1. Phosphate 8. Fumarate
2. Sodium 9. Sulfate
3. Potassium 10. Magnesium
4. Chloride 11. Calcium
5. Malae
6. Bromide
7. Nitrate
0,0 2,0 4,0 6,0 8,0 10,0 12,0 14,0 16,0 18,0 20,0
0,00
1,00
2,00
3,00
4,00
min
pA
20131030-12ions-runs #8 12ions-PFV1.5 evap50degs CAD_1
1
2
3
4
5
6 7
8 9
10
11
32. 32
Summary
• Selectivity is the KEY
• Mixed-mode chromatography addresses unmet challenges in
pharmaceutical analysis:
• API and counter ion by RP/AEX/CEX trimodal columns (e.g. Acclaim
Trinity P1 and P2)
• Unique Charged Aerosol Detector for consistent response of analytes
that are weak or non-chromophoric molecules
• Unmatched performance for counter ion analysis by dedicated column
technology, unique charged aerosol detection and established UltiMate
3000 technology
• New IonCount complete solution for ease of use and quick methods
development of new API and counter ion analysis
• Easy operation by predefined eWorkflows, Thermo Scientific™ Dionex™
Chromeleon™ 7.2 Chromatography Data System, and viper connection
tubing