Analysis of Pharmaceuticals Using IonExchange/Reversed-Phase Mixed-Mode Chromatography
Xiaodong Liu and Christopher Pohl
Overview
● Introduction
● Acclaim Trinity P1 column
● Nano-polymer Silica Hybrid (NSH) technology
● Features and values
● Pharmaceutical applications
● Pharmaceutical counterions (both anions and cations)
● Drug substances and counterions
● Mixture of acidic and basic drugs with respective counterions
● Conclusion
2
HPLC is an Important Tool for Determination of
Pharmaceutical Counterions
● Salt formation is a critical step in drug development
● ~ 50% of all drug molecules are administered as salts
● Benefits
● Improved biopharmaceutical properties (e.g. drug efficacy, permeability, controlled release, etc)
● Improved physicochemical properties (e.g. solubility, thermal stability, photo stability,
hygroscopicity, etc)
● Ease of purification and handling
● Extended patent protection
● HPLC is an important tool for determination of active pharmaceutical
ingredient (API) and counterions
● Counterion identity and stoichoimetry confirmation
● Combined with active pharmaceutical ingredient (API) result, ensure the safety,
identity, strength, purity and quality of the drug
3
Selectivity is the Key
N
 k 
  1
Rs 

4
 k  1
Rs – Resolution
N – Theoretical plates
α – Selectivity
k – Retention factor
Example:
To separate two analytes on a 5-m,150 mm C18 column
Assuming N = 10,000 plates/column, k >> 1, and α = 1.01  Rs = 1
Objective: increase Rs to 2
Approach 1 – increase N
needs 400% increase or N = 40,000 plates
Approach 2 – increase α
requires only 1% increase or α = 1.02
4
Mixed-Mode Stationary Phases
● Definition
● Hydrophobic interaction + ion-exchange interaction
● Classification
● Anion-exchange/reversed-phase
● Cation-exchange/reversed-phase
● Amphoteric or Zwitterionic/reversed-phase
● Values
● Adjustable selectivity
● Simplified mobile phase (no ion-pairing reagents)
● Simultaneous separation of different types of analytes
5
Mixed-Mode Column Chemistries
Silica Gel
I. Blend Packing
Hypersil Duet C18/SAX
(Thermo-Fisher Scientific)
II. Mixed Bonding
Alltech Mixed-Mode
(Grace)
Silica Gel
III. “Embedded”
Primesep Columns
(SIELC)
Silica Gel
IV. “Tipped”
Acclaim Mixed-Mode Columns
(Dionex)
RP – BLUE; IEX – RED
6
Dionex Acclaim Mixed-Mode Columns
Acclaim Mixed-Mode WAX-1
R
RR
N
R R R R RR R
N
N
N
N
Acclaim Mixed-Mode WCX-1
OOO- OOO C O C O C O CO C
Silica Gel
Silica gel:
Particle size:
Surface area:
Pore size:
7
Silica Gel
high-purity, porous, spherical
3 or 5 μm
300 m2/g
120 Å
Acclaim Mixed-Mode HILIC-1
OH OH OH OH OH
OH OH OH OH OH
Silica Gel
Separation Challenges
●
●
●
●
Pharmaceutical counterions (both anions and cations)
Drug substance and respective counterion (regardless of hydrophobicity)
Mixtures of basic and acidic drugs with respective counterions
Flexibility in method development
● Need for an “ideal” column that concurrently provides:
● Reversed-phase
● Anion-exchange
● Cation-exchange
8
Acclaim Trinity P1 – Column Chemistry
● High-purity, spherical, porous silica substrate
● Nano-polymer Silica Hybrid (NSH) technology
● Silica particles coated with nano-polymer beads
● Inner-pore: RP/WAX
● Outer surface: SCX
9
Acclaim Trinity P1 – Features, Values & Applications
● Features
● Multi-mode retention mechanism: RP, AEX, and CEX
● Adjustable selectivity
● Values
● Optimal selectivity and greater flexibility in method development
● Retain ionic and ionizable compounds without ion-pairing reagents
● A broad range of applications
● Applications
● Pharmaceutical counterions (both anions and cations)
● Drug substance and respective counterion (regardless of hydrophobicity)
● Mixtures of basic and acidic drugs with respective counterions
10
Acidic API and Counterion – pH Effect
400
2
1
pH4.1
mV
2
Column:
Dimensions:
Mobile Phase:
Temperature:
Flow Rate:
Inj. Volume:
Detection:
Sample:
Peaks:
Acclaim Trinity P1, 3 µm
3.0 x 50 mm
75/25 v/v CH3CN/30 mM (total) NH4OAc buffer
30 °C
0.5 mL/min
2.5 µL
ELS detector
Na, Naproxen (0.5 mg/mL in mobile phase)
1. Na+
2. Naproxen
1
ONa
t0
pH5.2
0
11
1
2
Minutes
3
4
Acidic API and Counterion – Ionic Strength Effect
400
1
2
20 mM NH4OAc
1
mV
30 mM NH4OAc
0
0
12
1
2
Minutes
Acclaim® Trinity P1, 3 µm
3.0 x 50 mm
75/25 v/v CH3CN/ NH4OAc buffer, pH 4.1
30 °C
0.5 mL/min
2.5 µL
ELS detector
Na, Naproxen (0.5 mg/mL in mobile phase)
1. Naproxen
2. Na+
2
t0
Column:
Dimensions:
Mobile Phase:
Temperature:
Flow Rate:
Inj. Volume:
Detection:
Sample:
Peaks:
3
4
ONa
Basic API and Counterion – Ionic Strength Effect
3000
1
20 mM NH4OAc (total)
2
1
30 mM NH4OAc (total)
mV
1. 1,1-Dimethylbiguanide
2. Chloride
1
40 mM NH4OAc (total)
t0
2
0
13
1
Acclaim® Trinity P1, 3 µm
3.0 x 50 mm
20/80 v/v CH3CN/ NH4OAc buffer, pH5.2
30 °C
0.5 mL/min
2.5 µL
ELS detector
1,1-Dimethylbiguanide•HCl
(0.2 mg/mL in mobile phase)
Peaks:
2
0
Column:
Dimensions:
Mobile Phase:
Temperature:
Flow Rate:
Inj. Volume:
Detection:
Sample:
2
Minutes
3
4
5
Penicillin G Potassium – Solvent Effect
2000
Column:
Dimensions:
Mobile Phase:
Temperature:
Flow Rate:
Inj. Volume:
Detection:
Sample:
2
1
90% CH3CN (v/v)
2
1
80% CH3CN (v/v)
mV
2
Peaks:
1. K+
2. Penicillin G
1
70% CH3CN (v/v)
2
60% CH3CN (v/v)
0
14
1
1
2
Minutes
Acclaim Trinity P1, 3 µm
3.0 x 50 mm
CH3CN/20 mM (total) NH4OAc, pH5.2
30 °C
0.6 mL/min
2.0 µL
ELS detector
Penicillin G, Potassium Salt
(0.5 mg/mL in mobile phase)
3
4
Method Ruggedness – Variation in Solvent
400
Column:
Dimensions:
Mobile Phase:
Temperature:
Flow Rate:
Inj. Volume:
Detection:
Sample:
Peaks:
2
Rs = 7.2
1
76% CH3CN (v/v)
2
Rs = 7.6
mV
1
Acclaim Trinity P1, 3 µm
3.0 x 50 mm
CH3CN/30 mM (total) NH4OAc buffer
30 °C
0.6 mL/min
2.0 µL
ELS detector
Na, Naproxen (0.5 mg/mL in mobile phase)
1. Na+
2. Naproxen
75% CH3CN (v/v)
2
1
t0
0
15
1
ONa
Rs = 8.6
74% CH3CN (v/v)
2
Minutes
3
4
Method Ruggedness – Variation in Buffer Concentration
400
Column:
Dimensions:
Mobile Phase:
Temperature:
Flow Rate:
Inj. Volume:
Detection:
Sample:
Peaks:
2
Rs = 7.8
1
32 mM NH4OAc
2
Rs = 8.2
1
mV
Acclaim Trinity P1, 3 µm
3.0 x 50 mm
75/25 v/v CH3CN/NH4OAc buffer
30 °C
0.6 mL/min
2.0 µL
ELS detector
Na, Naproxen (0.5 mg/mL in mobile phase)
1. Na+
2. Naproxen
30 mM NH4OAc
2
t0
0
16
1
1
ONa
Rs = 8.6
28 mM NH4OAc
2
Minutes
3
4
Isocratic Separation of Pharmaceutical Counterions
400
Column:
Dimensions:
Mobile Phase:
Temperature:
Flow Rate:
Inj. Volume:
Detection:
Peaks:
2
mV
34
5
6
7
1
8
9
10 11
0
0
17
3
6
Minutes
9
12
15
Acclaim Trinity P1, 3 µm
3.0 x 100 mm
60/40 v/v CH3CN/20 mM (total) NH4OAc, pH5
30 °C
0.5 mL/min
5 µL
ELS detector
(100 to 150 ppm)
1. Procaine
2. Choline
3. Tromethamine
4. Sodium
5. Potassium
6. Meglumine
7. Mesylate
8. Nitrate
9. Chloride
10. Bromide
11. Iodide
Gradient Separation of Pharmaceutical Counterions
1100
Column:
Dimensions:
Mobile Phase:
Gradient:
Time (min)
A
B
C
Temperature:
Flow Rate:
Inj. Volume:
Detection:
Peaks:
4
mV
10
9
2
1
3 5
7
6
8
1112 13
14
0
0
18
3
6
Minutes
9
15
16
12
15
Acclaim Trinity P1, 3 µm
3.0 x 50 mm
A - CH3CN, B - D.I. H2O, C – 0.2 M NH4OAc, pH4
-10
0
2
7
15
60
60
60
10
10
35
35
35
0
0
5
5
5
90
90
30 °C
0.5 mL/min
5 µL
ELS detector
(80 to 150 ppm)
1. Procaine
9. Chloride
2. Choline
10. Bromide
3. Tromethamine
11. Iodide
4. Sodium
12. Phosphate
5. Potassium
13. Malate
6. Meglumine
14. Tartrate
7. Mesylate
15. Citrate
8. Maleate
16. Oxalate
Fast Analysis – Na, Naproxen
500
2
t0
1.5 mL/min
1
2
0.5 mL/min
mV
ONa
t0
19
Acclaim Trinity P1, 3 µm
3.0 x 50 mm
75/25 v/v CH3CN/30 mM (total) NH4OAc, pH5
30 °C
0.5 and 1.5 mL/min
2.5 µL
ELS detector
Na, Naproxen (0.5 mg/mL in mobile phase)
1. Na+
2. Naproxen
1
0
Column:
Dimensions:
Mobile Phase:
Temperature:
Flow Rate:
Inj. Volume:
Detection:
Sample:
Peaks:
1
2
Minutes
3
4
Fast Analysis – Trimipramine Maleate
300
Column:
Dimensions:
Mobile Phase:
Temperature:
Flow Rate:
Inj. Volume:
Detection:
Sample:
Peaks:
1
1.8 mL/min
t0
2
1
mAU
1. Trimipramine
2. Maleate
0.5 mL/min
t0
0
20
2
1
2
Minutes
3
Acclaim Trinity P1, 3 µm
3.0 x 50 mm
30/70 v/v CH3CN/60 mM (total) NH4OAc, pH5
30 °C
0.5 and 1.8 mL/min
2 µL
UV, 254 nm
Trimipramine maleate (0.5 mg/mL in mobile phase)
4
Penicillin G Potassium
500
Column:
Dimensions:
Mobile Phase:
Temperature:
Flow Rate:
Inj. Volume:
Detection:
Sample:
2
mV
Acclaim Trinity P1, 3 µm
3.0 x 50 mm
60/40 CH3CN/20 mM (total) NH4OAc, pH5.2
30 °C
0.6 mL/min
2.0 µL
ELS detector
Penicillin G, Potassium Salt
(0.5 mg/mL in mobile phase)
Peaks:
1
0
21
1
1. K+
2. Penicillin G
2
Minutes
3
4
1,1-DimethylbigunideHCl
Column:
Dimensions:
Mobile Phase:
Temperature:
Flow Rate:
Inj. Volume:
Detection:
Sample:
800
1
mV
Acclaim® Trinity P1, 3 µm
3.0 x 50 mm
20/80 v/v CH3CN/ 30 mM NH4OAc,pH5.2
30 °C
0.5 mL/min
2.5 µL
ELS detector
1,1-Dimethylbiguanide•HCl
(0.2 mg/mL in mobile phase)
Peaks:
1. 1,1-Dimethylbiguanide
2. Chloride
t0
2
0
0
22
1
2
Minutes
3
4
5
Over-The-Counter Medicine – Advil ALLERGY and SINUS
600
4
mAU
t0
2
1
3
0
0
23
1
2
Minutes
3
4
Column:
Acclaim Trinity P1, 3 µm
Dimensions: 3.0 x 50 mm
Mobile Phase:A: CH3CN
B: D.I. H2O
C: 0.2 M NH4OAc, pH4.1
Gradient:
Time (min)
–4 0 0.1 1 4
A: 25 25 25 80 80
B: 65 65 65 0 0
C: 10 10 10 20 20
Temperature: 30 °C
Flow Rate:
0.5 mL/min
Inj. Volume: 2 µL
Detection:
UV, 254 nm
Peaks:
1. Pseudo-ephedrine
2. Chlorpheniramine
3. Maleate
4. Ibuprofen
* Background subtraction applied
Conclusion
● Acclaim Trinity P1 column
● High-purity silica column designed for pharmaceutical applications
● Concurrent reversed-phase, anion-exchange and cation-exchange properties
● Optimized selectivity for separating APIs and counterions
● Nano-polymer Silica Hybrid (NSH) technology – Optimal Selectivity
● Broad range of applications
●
●
●
●
●
Pharmaceutical counterions (both anions and cations)
Drug substance and respective counterion (regardless of hydrophobicity)
Mixtures of basic and acidic drugs with respective counterions
Mixture of acidic, basic and neutral active pharmaceutical ingredients (APIs)
……
ISO Slide Number
24
Thank You!
25