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©2009 Waters Corporation | COMPANY CONFIDENTIAL
LC Method Development in Bioanalysis
Successfully Developing
HILIC Methods
©2009 Waters Corporation | COMPANY CONFIDENTIAL
Challenges in Bioanalysis:Business Impact
Throughput
— Ranking of compound in discovery phase to ensure fast and accurate
decision-making
— Need to return results quickly to clinic
— Faster time to market
— Faster service for client
Productivity
— Cost-effective use of time and assets
— Maximize use of analyst’s time
— Accessible technology
o Usable by anyone
o Yields consistent high quality data
Right first time
— Reduce the occurrence of questionable results
— Reduce the occurrence of transcription errors
— Carry out sufficient experiments to understand both analytes and matrix
©2009 Waters Corporation | COMPANY CONFIDENTIAL
Matrix Effects
— Recent Crystal City meeting provided guidance on the
determination and acceptability of matrix effects
as a result of sample preparation
Incurred Sample Reanalysis (ISR)
— Recommendation that a subset of patient samples are
re-analyzed and compared to original
— Direct impact on productivity
— Increased costs
Metabolite Identification in Safety Testing (MIST)
— Recent FDA guidelines require that all metabolites with an
exposure >10% of the active must be quantified and identified
Challenges in Bioanalysis:Regulatory
©2009 Waters Corporation | COMPANY CONFIDENTIAL
Sensitivity is required to accurately monitor drug candidate and metabolites— More potent New Chemical Entities (NCE), lower dosing levels,
smaller sample volumes, extended release medicines
High throughput is not just desirable, it is a necessity— Hundreds of samples analyzed that possess diverse
physiochemical properties
— Fast turnaround time from sample receipt to Pharmacokinetic (PK) report is critical
Method ruggedness and reliability is essential for the generation of an accurate PK profile— Co-eluting endogenous materials and metabolites can result in
reduced assay accuracy
Data quality must be maintained — Better, more informed decisions
Challenges in Bioanalysis:Scientific
©2009 Waters Corporation | COMPANY CONFIDENTIAL
Typical Approach
LC/MS/MS is the analytical platform of choice
— Multiple Reaction Monitoring (MRM) provides a high degree of
selectivity
— Belief that MRM minimizes the impact on MS response from
endogenous materials or metabolites
Compromises in sample preparation and chromatography
are often made due to MS/MS selectivity
— Sample preparation
o Minimal use of solid phase extraction (SPE) for sample preparation
due to cost and method development time
o Protein precipitation (PPT) is often the sample preparation method
of choice
— Chromatography
o Universal reversed-phase (RP) methods employed
o Rapid gradients used with short (20 – 50 mm) C18 columns
©2009 Waters Corporation | COMPANY CONFIDENTIAL
Process for Developing a Successful Bioanalytical Method
Develop MRM Method
— Mass Spectrometry instrument platform
— Conventional procedure
— Consideration of parameters requiring optimization
— Novel approach to automated MRM method development
Develop LC Method
— LC instrument platform
— LC column choice
— Basic approach to fast method development
— Focused gradient method optimization
Assessment of Method as ―Fit for Purpose‖
— Sensitivity
— Specificity/matrix interference
— Additional steps?
©2009 Waters Corporation | COMPANY CONFIDENTIAL
Process for Developing a Successful Bioanalytical Method
Develop MRM Method
— Mass Spectrometry instrument platform
— Conventional procedure
— Consideration of parameters requiring optimization
— Novel approach to automated MRM method development
Develop LC Method
— LC instrument platform
— LC column choice
— Basic approach to fast method development
— Focused gradient method optimization
Assessment of Method as ―Fit for Purpose‖
— Sensitivity
— Specificity/matrix interference
— Additional steps?
©2009 Waters Corporation | COMPANY CONFIDENTIAL 8
Column Selection in Method Development
BEH C18
BEH C8
BEH Phenyl
BEH Shield RP18
BEH HILIC
BEH Amide
BEH 300Å C18
BEH 300Å C4
HSS T3
HSS C18
HSS C18 SB
Three particle substrates [130Å and 300Å BEH, HSS]
— All are available in HPLC and UPLC particle sizes
Wide and growing selection of column chemistries
— 11 stationary phases
o BEH 130Å C18, C8, Shield RP18, Phenyl, HILIC, Amide
o BEH 300Å C18 and C4
o HSS C18, T3, C18 SB
Proven application-based solutions
— AAA, OST, PST, PrST and Glycan
Hybrid Particle Technology
— Widest pH range for easier method development
— Increased mass loading / loadability
— Pressure tolerance
— Choice of 130Å (small molecules) & 300Å (large molecules)
pore sizes
Scalability with XBridge and HSS HPLC columns
VanGuard Pre-columns
eCord Technology
©2009 Waters Corporation | COMPANY CONFIDENTIAL 9
Benefits of High pH Mobile Phase in Mass Spectrometry
1. 9-OH Risperidone
2. Risperidone
3. Clozapine (I.S.)
Low pH High pH
MeOH
ACN
Evaluate peak shapes, resolution and sensitivity
Time0.50 1.00 1.50 2.00 2.50
%
0
0.50 1.00 1.50 2.00 2.50
%
0
2.86e6
1.11
1.06
1.25
2.86e6
0.880.84
0.95
Time0.50 1.00 1.50 2.00 2.50
%
0
0.50 1.00 1.50 2.00 2.50
%
0
2.86e6
1.11
1.06
1.25
2.86e6
0.880.84
0.95
Time0.50 1.00 1.50 2.00 2.50
%
0
0.50 1.00 1.50 2.00 2.50
%
0
2.86e6
Time0.50 1.00 1.50 2.00 2.50
%
0
0.50 1.00 1.50 2.00 2.50
%
0
2.86e6
1.11
1.06
1.25
2.86e6
1.11
1.06
1.25
2.86e6
0.880.84
0.95
Time0.50 1.00 1.50 2.00 2.50
%
0
0.50 1.00 1.50 2.00 2.50
%
0
2.86e61.42
1.35
1.54
2.86e61.04
0.98
1.26
Time0.50 1.00 1.50 2.00 2.50
%
0
0.50 1.00 1.50 2.00 2.50
%
0
2.86e61.42
1.35
1.54
2.86e61.04
0.98
1.26
Time0.50 1.00 1.50 2.00 2.50
%
0
0.50 1.00 1.50 2.00 2.50
%
0
2.86e6
Time0.50 1.00 1.50 2.00 2.50
%
0
0.50 1.00 1.50 2.00 2.50
%
0
2.86e61.42
1.35
1.54
2.86e6
1.42
1.35
1.54
2.86e61.04
0.98
1.26
Independent of which organic modifier is used, these basic compounds exhibit a higher MS
response in a high pH mobile phase
Why?
pH 3 pH 10
©2009 Waters Corporation | COMPANY CONFIDENTIAL 10
Benefit of High pH Separations:Enhanced Retention of Basic Compounds
pH 10
Nortriptyline Amitriptyline
Minutes0 1 2 3
0 1 2 3
pH 8 Nortriptyline
Amitriptyline
0 1 2 3
pH 2
0
10
20
30
40
0 1 2 3 4 5 6 7 8 9 10 11 12
8
Amitriptyline
Nortriptyline
k
pH
2
10
Silica-based materials dissolve at high pH
N
CH3
CH3
NH
CH3
Analytes retain more when they are
un-ionized
©2009 Waters Corporation | COMPANY CONFIDENTIAL 11
LC Column and Mobile Phase Considerations
Choosing an LC column that has extended pH
utility (1-12) allows the flexibility to investigate
analyte response
—Manipulate retention of acids and bases
— Improve the retention of polar metabolites
—Elution in higher organic concentration leads to improved
desolvation in the MS source
—Analytes eluting in their un-ionized form combined with
proper choice of mobile phase buffer/concentration can
lead to significant increase in MS response*
* R Kostiainen, T.J Kauppila, J Chromatogr A (2009), 1216, 685-699
©2009 Waters Corporation | COMPANY CONFIDENTIAL 12
Wall
Surface
OSi
OSi
OSi
OSi
OSi
OSi
OSi
OSi
OSi
O
OH OH OH OH OH CH2 CH2OH OH
O O O O O O O
OSi
O
SiO
Si
O
SiO
SiO
Si Si Si SiO
CH2
CH2O O O O O O
Si
SiO
O
O
OH
O
CH2
CH2
CH2
CH2
OSi
OSi
OSi
OSi
OSi
OSi
OSi
OSi
OSi
O
CH2
CH2
OOOOOOO
Si
OCH2
CH2
O
OO
2nd Generation Hybrid Particles:Ethylene Bridged Hybrid (BEH) Particles
ReducedSilanols
HydrophobicGroup
Bridging Group
Hybrid Particle Attribute
Surface hybrid groups reduce surface silanol concentration
Internal bridging groups provide high interconnectivity
Internal hybrid groups provide hydrophobicity
Benefit
Improved USP tailing factors
Increased chemical and mechanical stability
Increased high pH stability
©2009 Waters Corporation | COMPANY CONFIDENTIAL 13
Improved Retention and Sensitivity:High pH Mobile Phase
10-hydroxymorphine
Morphine-3ß–o-glucuronide
Morphine-6ß–o-glucuronide
Morphine N-oxide
6-acetylmorphine
Morphine
HIGH pH0.1% NH4OH
LOW pH0.1% HCOOH
Compounds
1. 10-hydroxymorphine
2. Morphine-3β-D-glucuronide
3. Morphine-6β-D-glucuronide
4. Morphine
5. Morphine N-oxide
6. 6-acetylmorphine
©2009 Waters Corporation | COMPANY CONFIDENTIAL 14
BEH Technology in Bioanalysis:High pH Case Study
Mobile phase pH can impact
retention, selectivity and
sensitivity if the analytes are
ionizable
Selectively elute analytes away
from phospholipids
Basic analytes are unionized at
high pH, and therefore, retain
greater on the stationary
phase, eluting in higher organic
E. Chambers et al. J. Chromatogr. B 852 (2007) 22 - 34
CH3
CH3
CH3
CH3 N+
O
OH
O-
O
O
P
H
OO
Phospholipid: 1-Stearoyl-2-Hydroxy-sn-Glycero-3-Phosphocholine
Molecular Weight: 523.34Molecular Formula: C26H54NO7P
©2009 Waters Corporation | COMPANY CONFIDENTIAL 15
BEH Technology in Bioanalysis:High pH Case Study
E. Chambers et al. J. Chromatogr. B 852 (2007) 22 - 34
pH 2.7 pH 9
2.3X Response
1.6X Response
Column: ACQUITY UPLC® BEH C18, 2.1 x 50 mm 1.7 µm
©2009 Waters Corporation | COMPANY CONFIDENTIAL 16
BEH Technology in Bioanalysis:High pH Case Study Summary
E. Chambers et al. J. Chromatogr. B 852 (2007) 22 - 34
Benefits:
Higher sensitivity
—Abundant protonated molecules can be produced in high pH conditions even though basic analytes are un-ionized
—Greater retention for un-ionized molecules, resulting in elution in a higher organic mobile phase (easier desolvation)
—Selectivity can be manipulated to selectively elute analytes away from residual phospholipids
—Highly efficient, symmetrical peaks resulting in narrow chromatographic bands
©2009 Waters Corporation | COMPANY CONFIDENTIAL 17
Typical UPLC® Method Scouting Conditions
ACQUITY UPLC® BEH C18, 2.1 x 50 mm, 1.7 µm—Wide pH range (1-12)
— Lower silanol activity than silica
Low and high pH mobile phases
—0.1% Formic Acid (pH ~2.7)
—0.1% Ammonium Hydroxide in H2O (pH ~11.3)
Two organic modifiers
—Methanol and acetonitrile
Generic gradient: 2% to 98% B in 2 min
Best chromatography chosen as a starting point for optimization
—Good peak shapes
—Sensitivity
—Resolution/selectivity
©2009 Waters Corporation | COMPANY CONFIDENTIAL 18
Alprazolam LC Screening
High pH
ACN
MeOH
Low pHAlprazolam is a very weak base and exhibits very little change in retention at the different pHs
Note the significant difference in MS sensitivity when the molecule is analyzed under high pH mobile phase conditions
MeOH is a weaker eluting solvent resulting in increased retention of the analyte
©2009 Waters Corporation | COMPANY CONFIDENTIAL
Retention and Sensitivity Challenges:Polar Compounds and Metabolites
Polar Compounds and Metabolites:
Often overlooked due to their elution in the void space of
the column
Co-elute with endogenous species present within biological
matrices, adversely effecting assay precision and accuracy
Require high aqueous mobile phases that may adversely
effect MS response
Ion-pairing reagents aid in retention, however, often
suppress MS response
©2009 Waters Corporation | COMPANY CONFIDENTIAL 20
What is HILIC?
HILIC - Hydrophilic Interaction Chromatography
—Term coined in 1990 to distinguish from normal-phase*
HILIC is a variation of normal-phase chromatography without the disadvantages of using solvents that are not miscible in water
—“Reverse reversed-phase” or “aqueous normal-phase” chromatography
Stationary phase is a POLAR material
—Silica, hybrid, cyano, amino, diol, amide
The mobile phase is highly organic (> 80% ACN) with a smaller amount of aqueous mobile phase
—Water (or the polar solvent(s)) is the strong, eluting solvent
*Alpert, A. J. J.Chromatogr. 499 (1990) 177-196.
©2009 Waters Corporation | COMPANY CONFIDENTIAL 21
Benefits of HILIC
Retention of highly polar analytes not retained by reversed-phase
—Less interference from non-polar matrix components
Complementary selectivity to reversed-phase
—Polar metabolites/impurities/degradants retain more than parent compound
Enhanced sensitivity in mass spectrometry
—High organic mobile phases (> 80% ACN) promotes enhanced ESI-MS response
—Direct injection of PPT supernatant without dilution
—Facilitates use of lower volume samples
Improved sample throughput
—Direct injection of high organic extracts from PPT, LLE or SPE without the need for dilution or evaporation and reconstitution
©2009 Waters Corporation | COMPANY CONFIDENTIAL
HILIC-MS/MS Sensitivity
Time0.40 0.80 1.20 1.60 2.00 2.40 2.80 3.20 3.60
MRM of 5 Channels ES+
180.2 > 148.2
3.01e4
x150 1.66
0.178 nmol/L metanephrine
LLOQ (S:N ≥ 10) calculated by extrapolation = 0.012 nmol/L
Time0.40 0.80 1.20 1.60 2.00 2.40 2.80 3.20 3.60
MRM of 5 Channels ES+
166.2 > 134.2
2.11e4
x901.67
0.525 nmol/L normetanephrineLLOQ (S:N ≥ 10) calculated by
extrapolation = 0.06 nmol/L
O NH
OH
OH
CH3CH3
O
OH
NH2
OHCH3
metanephrine
normetanephrine
Direct injection of SPE Eluate
©2009 Waters Corporation | COMPANY CONFIDENTIAL 23
When To Use HILIC
When to Use HILIC:
Need improved retention of
hydrophilic or ionizable
compounds
Need improved MS
response for polar or
ionizable compounds
Need improved sample
throughput for assays using
organic extraction
Reversed-phase
polar non-polar
Compound Index
Normal-phase
ESI-
MS
Re
spo
nse
exce
llen
tp
oo
r
HILIC
©2009 Waters Corporation | COMPANY CONFIDENTIAL 24
Outline
Overview of HILIC
Retention mechanisms and characteristics
—Retention mechanisms
—Retention and selectivity matrix
o Organic modifier
o Stationary phase
o Mobile phase pH
Practical considerations
HILIC method development strategy
Conclusions
©2009 Waters Corporation | COMPANY CONFIDENTIAL 25
Multi-modal Retention Mechanisms in HILIC
Combination of partitioning, ion-exchange
and hydrogen bonding
• Polar analyte partitions between bulk mobile phase and partially immobilized polar layer on material surface
• Secondary interactions between surface silanols and/or functional groups with the charged analyte leading to ion-exchange
• Hydrogen bonding between positively charged analyte and negatively charged surface silanols
D.V. McCalley, U. D. Neue, J. Chromatogr. A 1192 (2008) 225-229
E.S. Grumbach, D.M. Diehl, U.D. Neue, J. Sep. Sci. 31 (2008), 1511-1518
A. Méndez, E. Bosch, M. Rosés, U. D. Neue, J. Chromatogr. A 986 (2003), 33-44
©2009 Waters Corporation | COMPANY CONFIDENTIAL 26
Influence of Organic Solvent Composition on Retention
McCalley, D.V., J. Chromatogr. A, 2007, 1171, 46-55
0
2
4
6
8
10
12
14
16
5 10 20 30 40 50 60 70 80 90 95
% ACN in mobile phase
Ret
en
tio
n F
acto
r (k
)
Hydrophobic retention on siloxane bonds
Liquid-liquid partitioningNicotinic acid
pKa = 2.2, 4.8
Nortriptyline pKa = 2.2, 4.8
©2009 Waters Corporation | COMPANY CONFIDENTIAL 27
Retention and Selectivity Matrix
HILIC Retention and
Selectivity
Stationary Phase
Organic Modifier
Mobile Phase pH
©2009 Waters Corporation | COMPANY CONFIDENTIAL 28
Retention and Selectivity Matrix
HILIC Retention and
Selectivity
Stationary Phase
Organic Modifier
Mobile Phase pH
©2009 Waters Corporation | COMPANY CONFIDENTIAL 29
Influence of % Acetonitrile on Retention
A
B
0
2
4
6
8
10
12
14
16
70 75 80 85 90 95
Rete
nti
on
Facto
r
% Acetonitrile
Silica Particle
0
2
4
6
8
10
12
14
16
70 75 80 85 90 95
Rete
nti
on
Facto
r
% Acetonitrile
BEH Particle
Nicotinic acid
Nortriptyline
Cytosine
Methacrylic acid
Nicotinic acid
Nortriptyline
Cytosine
Methacrylic acid
A
B
0
2
4
6
8
10
12
14
16
70 75 80 85 90 95
Rete
nti
on
Facto
r
% Acetonitrile
Silica Particle
0
2
4
6
8
10
12
14
16
70 75 80 85 90 95
Rete
nti
on
Facto
r
% Acetonitrile
BEH Particle
Nicotinic acid
Nortriptyline
Cytosine
Methacrylic acid
Nicotinic acid
Nortriptyline
Cytosine
Methacrylic acid
Exponential retention increase with mobile phases containing more than 90% acetonitrile
O
CH2OH
CH3
Methacrylic Acid pKa 4.58
N
NH
O
NH2
CytosinepKa = 12.2
NHCH3
NortriptylinepKa = 10
O
N
OH
Nicotinic AcidpKa = 2.2, 4.8
©2009 Waters Corporation | COMPANY CONFIDENTIAL 30
Weakest
Strongest
Use a less polar solvent toIncrease retentionof polar analytes
Primary
[Weak] Solvents
Acetone
Acetonitrile
Isopropanol
Ethanol
Methanol
Water
Elution
[Strong] Solvents
Solvent Selectivity and Elution Strength
©2009 Waters Corporation | COMPANY CONFIDENTIAL 31
Influence of Polar Modifier on Retention and Selectivity
10 mM ammonium acetate with 0.02% acetic acid
Analytes:1: methacrylic acid 2: cytosine 3: nortriptyline 4: nicotinic acid
12 3
4
12
3
4
1 23
4
1
34
90:10 ACN:H2O
90:5:5 ACN:H2O:MeOH
90:5:5 ACN:H2O:EtOH
90:5:5 ACN:H2O:IPA
Minutes
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
2
Retention increases with decreasing solvent polarity
©2009 Waters Corporation | COMPANY CONFIDENTIAL 32
Retention and Selectivity Matrix
HILIC Retention and
Selectivity
Stationary Phase
Organic Modifier
Mobile Phase pH
©2009 Waters Corporation | COMPANY CONFIDENTIAL 33
Stationary Phases for HILIC separations
ACQUITY UPLC® BEH AmideXBridgeTM Amide
ACQUITY UPLC® BEH HILICXBridgeTM HILIC
Atlantis® HILIC Silica
Silica HILIC Columns(pH range 1 – 5)
Hybrid HILIC Columns(pH range 1 – 11)
©2009 Waters Corporation | COMPANY CONFIDENTIAL 34
Influence of Stationary Phase on Retention
ACQUITY UPLC BEH HILIC 2.1 x 50 mm, 1.7 µm
Unbonded hybrid with low silanol activity
4 5
Minutes
12
3
3
1 24
5
1 23
4
5
0 1 2 3
ACQUITY UPLC BEH Amide 2.1 x 50 mm, 1.7 µm
Bonded hybrid
Atlantis HILIC Silica 2.1 x 50 mm, 3 µm
Unbonded silica with high silanol activity
(1) acenaphthene (2) thymine (3) 5-fluoroorotic acid (4) adenine (5) cytosine; UV 254 nm
©2009 Waters Corporation | COMPANY CONFIDENTIAL 35
Influence of Stationary Phase on Selectivity
0
5
10
15
20
25
60 70 80 90 95
Re
ten
tio
n F
acto
r
% Acetonitrile
ACQUITY UPLC BEH HILIC
nicotinic acid
methacrylic acid
nortriptyline
cytosine
0
5
10
15
20
25
60 70 80 90 95
Re
ten
tio
n F
acto
r
% Acetonitrile
ACQUITY UPLC BEH Amide
nicotinic acid
methacrylic acid
nortriptyline
cytosine
O
CH2OH
CH3
Methacrylic Acid pKa 4.58
N
NH
O
NH2
CytosinepKa = 12.2
NHCH3
NortriptylinepKa = 10
O
N
OH
Nicotinic AcidpKa = 2.2, 4.8
Stationary phase functional groups can influence retention
©2009 Waters Corporation | COMPANY CONFIDENTIAL 36
Retention and Selectivity Matrix
HILIC Retention and
Selectivity
Stationary Phase
Organic Modifier
Mobile Phase pH
©2009 Waters Corporation | COMPANY CONFIDENTIAL 37
Influence of Mobile Phase pH on Retention and Selectivity
Minutes0.00 1.00 2.00 3.00
1
2
3
4
pH 3
pH 9
Compounds1. Methacrylic acid2. Nortriptyline3. Nicotinic acid4. cytosine
1
2
3
4
ACQUITY UPLC BEH Amide, 2.1 x 50 mm , 1.7 µm
O
CH2OH
CH3
Methacrylic Acid pKa 4.58
N
NH
O
NH2
CytosinepKa = 12.2
NHCH3
NortriptylinepKa = 10
O
N
OH
Nicotinic AcidpKa = 2.2, 4.8
©2009 Waters Corporation | COMPANY CONFIDENTIAL 38
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
1
2
4
36
5
87
Compounds1. Nicotinamide2. Pyridoxine3. Riboflavin4. Nicotinic acid5. Thiamine6. Ascorbic Acid7. B128. Folic Acid
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
pH 3
pH 91
2
3
45
6
78
O
N
OH
Nicotinic acid
O
OH OH
O
OH
OH
Ascorbic acid
N
NH
N
N
NH
O
O
O
NH2
NH
O
OHOH
Folic acid
Minutes
Influence of Mobile Phase pH on Retention and Selectivity
ACQUITY UPLC BEH Amide, 2.1 x 50 mm , 1.7 µm
©2009 Waters Corporation | COMPANY CONFIDENTIAL 39
Influence of Mobile Phase pH on MS Signal Intensity
1.00E+05 1.00E+06 1.00E+07 1.00E+08 1.00E+09 1.00E+10
Uracil
Nicotinic acid
Thymine
5-fluorouracil
salicylic acid
4-aminosalicylic acid
2-naphthalenesulfonic acid
EMPA
IMPA
PMPA
CMPA
MMPA
Benzylamine
Cytosine
5-fluorocytosine
Adenine
acyclovir
Diphenhydramine
Nortriptyline
Thiamine
Procainamide
Morphine
Ion counts
pH 9
pH 3
ES-
ES+
©2009 Waters Corporation | COMPANY CONFIDENTIAL 40
Influence of Mobile Phase pH on MS Signal Intensity
High pHLow pH
ACQUITY UPLC BEH HILICInjection solvent 95/5 ACN/H2O + 0.2% HCOOH
Time0.50 1.00 1.50 2.00 2.50
0.50 1.00 1.50 2.00 2.50
0.50 1.00 1.50 2.00 2.50
MRM of 3 Channels ES+ 182.1 > 164
1.20e50.94
MRM of 3 Channels ES+ 172 > 153.9
1.20e5
0.94
MRM of 3 Channels ES+ 160 > 141.9
1.20e50.89
Time0.50 1.00 1.50 2.00 2.50
0.50 1.00 1.50 2.00 2.50
0.50 1.00 1.50 2.00 2.50
MRM of 3 Channels ES+ 182.1 > 164
1.20e5
MRM of 3 Channels ES+ 172 > 153.9
1.20e5
MRM of 3 Channels ES+ 160 > 141.9
1.20e5
pregabalin
gabapentin
gabapentin d10
pregabalin
gabapentin
gabapentin d10
©2009 Waters Corporation | COMPANY CONFIDENTIAL 41
Retention and Selectivity Summary
HILIC Retention and
Selectivity
Stationary Phase
Organic Modifier
Mobile Phase pH
•Decreasing solvent polarity increases retention
•Screen multiple columns to maximize retention and selectivity differences
•Analytes have greater retention when they are ionized [acids at high pH, bases at low pH]
©2009 Waters Corporation | COMPANY CONFIDENTIAL 42
Outline
Overview of HILIC
Retention mechanisms and characteristics
Practical considerations
—Common buffers and additives
—Mobile phase preparation
—Column equilibration
—Sample diluent
HILIC method development strategy
Conclusions
©2009 Waters Corporation | COMPANY CONFIDENTIAL 43
Before You Start:Common HILIC mobile phases
Common buffers/additives*
—Ammonium formate, ammonium acetate
—Formic acid, ammonium hydroxide, acetic acid
—Phosphate salt buffers ARE NOT recommended due to precipitation in the
highly organic mobile phase (phosphoric acid is OK)
Recommended buffer concentration: 10 – 20 mM ON-COLUMN
Recommended additive concentration: 0.2% ON-COLUMN
*The actual pH of the mobile phase may be 1 pH unit closer to neutral due to the highly organic mobile phaseCanals, I.; Oumada, F. Z.; Roses, M.; Bosch, E. J. Chromatogr. A. 911 (2001) 191-202.
Espinosa, S.; Bosch, E.; Roses, M. Anal. Chem. 72 (2000) 5193-5200.
©2009 Waters Corporation | COMPANY CONFIDENTIAL 44
AU
0.00
1.00
AU
0.00
1.00
AU
0.00
1.00
AU
0.00
1.00
AU
0.00
1.00
Minutes
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00
1,2 3 4
3
2
41
3
2
41
3
2
41
3
2
41
Effect of Buffer Concentration on Retention: pH 3.0
0 mMammonium
formate
2.5 mMammonium
formate
1. Methacrylic acid2. Nicotinic acid3. Nortriptyline4. Cytosine
All contain 90:10 MeCN:H2O
5.0 mMammonium
formate
10 mMammonium
formate
20 mMammonium
formate
O
CH2OH
CH3
Methacrylic Acid pKa 4.58
N
NH
O
NH2
CytosinepKa = 12.2
NHCH3
NortriptylinepKa = 10
O
N
OH
Nicotinic AcidpKa = 2.2, 4.8
©2009 Waters Corporation | COMPANY CONFIDENTIAL 45
Effect of Buffer Concentration on Retention:pH 9.0
0.00
1.00
0.00
1.00
0.00
1.00
0.00
1.00
0.00
1.00
Minutes
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00
3
1 2
4
3
13
4
2
1
3 4
2
3
1
4
4
2
1,2
AU
AU
AU
AU
AU
0 mMammonium
acetate
2.5 mMammonium
acetate
5.0 mMammonium
acetate
10 mMammonium
acetate
20 mMammonium
acetate
All contain 90:10 MeCN:H2O
O
CH2OH
CH3
Methacrylic Acid pKa 4.58
N
NH
O
NH2
CytosinepKa = 12.2
NHCH3
NortriptylinepKa = 10
O
N
OH
Nicotinic AcidpKa = 2.2, 4.8
1. Methacrylic acid2. Nicotinic acid3. Nortriptyline4. Cytosine
©2009 Waters Corporation | COMPANY CONFIDENTIAL 46
Additives vs. Buffered Mobile Phases:Impact on Retention and Peak Shape
1,2
43
3
1
4
2
Minutes
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00
0.2% ammonium hydroxide pH 9
20 mM ammonium acetate pH 9
Minutes
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00
3
2
4
1
2 4
1
20 mM ammonium formatepH 3
0.2% formic acid pH 33
1. Methacrylic acid2. Nicotinic acid3. Nortriptyline4. Cytosine
pH 9 Observations
Acids are unretained without a buffered mobile phase
Selectivity shifts for basic compounds
pH 3 Observations
Poor peak shape and retention for bases without a buffered mobile phase
Selectivity shifts for acidic compounds
All contain 90:10 MeCN:H2O
©2009 Waters Corporation | COMPANY CONFIDENTIAL 47
Influence of Buffer Concentration on Peak Shape
ACQUITY UPLC BEH HILICInjection solvent 75/25 ACN/MeOH + 0.2% HCOOHBuffer: ammonium formate (pH 3)
pregabalin
gabapentin
gabapentin d10
Time0.50 1.00 1.50 2.00 2.50
0.50 1.00 1.50 2.00 2.50
0.50 1.00 1.50 2.00 2.50
MRM of 3 Channels ES+ 182.1 > 164
1.28e50.95
MRM of 3 Channels ES+ 172 > 153.9
1.28e5
0.98
MRM of 3 Channels ES+ 160 > 141.9
1.28e50.93
pregabalin
gabapentin
gabapentin d10
Time0.50 1.00 1.50 2.00 2.50
0.50 1.00 1.50 2.00 2.50
0.50 1.00 1.50 2.00 2.50
MRM of 3 Channels ES+ 182.1 > 164
1.26e50.94
MRM of 3 Channels ES+ 172 > 153.9
1.26e5
MRM of 3 Channels ES+ 160 > 141.9
1.26e5
10 mM Buffer 20 mM Buffer
©2009 Waters Corporation | COMPANY CONFIDENTIAL 48
Before You Start:Mobile Phase Preparation
Additives
—Replace 0.2% of mobile phase volume with additive [2 mL out of 1 L]
Buffers
—Prepare a stock buffer [typically 200 mM] and then dilute 20-fold into
the running mobile phase [10 mM concentration on column]
—Example: Prepare stock of 200 mM ammonium formate, pH 3. For a
mobile phase containing 95% ACN and 5% water with 10 mM
ammonium formate, pH 3, add 50 mL of stock buffer to 950 mL of ACN.
For the best gradient performance and reproducibility, it is
recommended that the additive or buffer be added to both
aqueous and organic mobile phase bottles
©2009 Waters Corporation | COMPANY CONFIDENTIAL 49
Instrument Wash Solvents
— Strong needle wash: 9:1 acetonitrile: water
— Weak needle wash/purge solvent: initial mobile phase conditions [excluding salt,
additive or buffer]
Brand new column— Run 50 empty column volumes of 50:50 acetonitrile:water with 10 mM buffer or
0.2% additive solution
Column equilibration
— Equilibrate with 20 empty column volumes of initial mobile phase conditions
Gradient separations
— Re-equilibrate with 5 to 8 empty column volumes
As with any column, insufficient equilibration can cause drifting retention times
Before You Start: Column Equilibration and Wash Solvents
©2009 Waters Corporation | COMPANY CONFIDENTIAL 50
Sample diluent strongly influences solubility and peak shape (just like reversed-phase)
Sample diluent should be at least 75% acetonitrile or as close to initial mobile phase conditions as possible
However, polar analytes often have low solubilities in organic solvents
General purpose HILIC diluent—75:25 acetonitrile:methanol works for most polar analytes
—Offers a compromise between solubility and peak shape
—Adjust according to your analytes (add 0.2% formic acid to increase solubility)
— In some cases, 25% methanol may be too polar to use as an injection solvent
Before You Start:Influence of Sample Diluent
©2009 Waters Corporation | COMPANY CONFIDENTIAL 51
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00
100% H2O
50 ACN: 50 H2O
75 ACN: 25 H2O
S
12
34
S1
2
3 4
S
1 2
34
Peak shape improvesas % ACN in the
diluent increases.
What about alternative polar organic solvents?
ACQUITY UPLC® BEH HILIC2.1 x 100 mm, 1.7 µm
Analytes1. Methacrylic acid2. Cytosine 3. Nortriptyline 4. Nicotinic acid
Influence of Sample DiluentWater as the Polar Solvent
©2009 Waters Corporation | COMPANY CONFIDENTIAL 52
Influence of Sample DiluentMethanol as the Polar Solvent
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00
100% MeOH
50 ACN: 50 MeOH
75 ACN: 25 MeOH
S
12
34
S1
23
4
S
12
34
Peak shape and solubility improve by replacing water with methanol
Peak shape improvesas % ACN in the
diluent increases.
ACQUITY UPLC® BEH HILIC2.1 x 100 mm, 1.7 µm
Analytes1. Methacrylic acid2. Cytosine 3. Nortriptyline 4. Nicotinic acid
©2009 Waters Corporation | COMPANY CONFIDENTIAL 53
Influence of Sample Diluent on Sensitivity
pregabalin
gabapentin
gabapentin d10
Time0.50 1.00 1.50 2.00 2.50
0.50 1.00 1.50 2.00 2.50
0.50 1.00 1.50 2.00 2.50
MRM of 3 Channels ES+ 182.1 > 164
1.26e5
0.94
MRM of 3 Channels ES+ 172 > 153.9
1.26e5
0.94
MRM of 3 Channels ES+ 160 > 141.9
1.26e5
0.89
gabapentin
gabapentin d10
0.50 1.00 1.50 2.00 2.50
0.50 1.00 1.50 2.00 2.50
0.94
pregabalin
Time0.50 1.00 1.50 2.00 2.50
ACQUITY UPLC BEH HILICBuffer: ammonium formate (pH 3)
Sample Diluent: 75/25 ACN/MeOH + 0.2% HCOOHSample Diluent: 95/5 ACN/H2O + 0.2% HCOOH
©2009 Waters Corporation | COMPANY CONFIDENTIAL 54
Outline
Overview of HILIC
Retention mechanisms and characteristics
Practical considerations
HILIC method development strategy
—Method development screening approach
—Implementing the approach: practical example
—Optimization steps
Conclusions
©2009 Waters Corporation | COMPANY CONFIDENTIAL 55
Waters HILIC screening strategy
pH 3
ACQUITY UPLC® BEH HILIC2.1 x 50 mm, 1.7 µm
Op
tim
izat
ion
pH 9
ACQUITY UPLC® BEH Amide2.1 x 50 mm, 1.7 µm
Atlantis HILIC Silica2.1 x 50 mm, 3 µm
Where do I start?• Initial scouting gradient from 95 to 50% acetonitrile over 2 minutes• At least 5% should be a polar solvent (i.e., water or methanol)
©2009 Waters Corporation | COMPANY CONFIDENTIAL 56
Instrument: ACQUITY UPLC system with ACQUITY PDA and Xevo TQ MS
Columns: ACQUITY UPLC BEH Amide, 2.1 x 50 mm, 1.7 µm
ACQUITY UPLC BEH HILIC, 2.1 x 50 mm, 1.7 µm
Atlantis HILIC Silica, 2.1 x 50 mm, 3 µm
MP A1: 20 mM HCOONH4 and 0.125% HCOOH, pH 3.0*
MP B1: 95/5 ACN/H2O with 20 mM HCOONH4 and 0.125% HCOOH, pH 3.0*
MP A2: 20 mM CH3COONH4 and 0.04% NH4OH, pH 9.0*
MP B2: 95/5 ACN/H2O with 20 mM CH3COONH4 and 0.04% NH4OH, pH 9.0*
Gradient: 99.5% B to 50% B in 2 min, reset (total run time = 3 min)
Flow rate: 0.6 mL/min
Inj. vol.: 5 µL
Sample Diluent:75/25 ACN/MeOH
(0.2% HCOOH needed in some cases for solubility)
Column Temp.: 30°C
Strong and weak needle washes: 95/5 ACN/H2O
*pH of stock buffer at 200 mM in water
Automated Screening Conditions
©2009 Waters Corporation | COMPANY CONFIDENTIAL 57
Illustrating the Methods Development Approach
As we go through the data for each separation, we should
have some questions in mind to help select the best pH,
organic solvent and column
—Adequate retention/separation from matrix components?
— Is a gradient or isocratic method desired?
—Sensitivity requirements?
Step 1: Review data and select pH
Step 2: Review data and select column
Step 3: Optimize/fine-tune separation as necessary
©2009 Waters Corporation | COMPANY CONFIDENTIAL 58
Implementing the Approach:Example 1: Fentanyl
Fentanyl is a narcotic pain
medicine nearly 100x more
potent than morphine
Fentanyl is used to treat cancer
pain that is not controlled by
other medicines.
N
N
O
CH3
Fentanylm.w. 336.47
©2009 Waters Corporation | COMPANY CONFIDENTIAL 59
Stationary Phase Selectivity at Low pH:Fentanyl
pH 3 Observations
Poor retention and peak shape on BEH Amide and
HILIC
Good retention and peak shape on Atlantis HILIC
Silica
BEH Amide
BEH HILIC
Atlantis HILIC Silica
Time0.50 1.00 1.50 2.00 2.50
0.50 1.00 1.50 2.00 2.50
0.50 1.00 1.50 2.00 2.50
MRM of 2 Channels ES+ 337.15 > 188.05
7.20e60.27
MRM of 2 Channels ES+ 337.15 > 188.05
7.20e6
0.64
MRM of 2 Channels ES+ 337.15 > 188.05
7.20e6
0.94
N
N
O
CH3
Fentanylm.w. 336.47
©2009 Waters Corporation | COMPANY CONFIDENTIAL 60
Mobile Phase Selectivity and Sensitivity:Fentanyl
Low pH High pH
BEH Amide
BEH HILIC
BEH Amide
BEH HILICTime
0.50 1.00 1.50 2.00 2.50
0.50 1.00 1.50 2.00 2.50
MRM of 2 Channels ES+ 337.15 > 188.05
5.34e6
0.27
0.45
MRM of 2 Channels ES+ 337.15 > 188.05
5.34e6
0.64
Time0.50 1.00 1.50 2.00 2.50
0.50 1.00 1.50 2.00 2.50
MRM of 2 Channels ES+ 337.15 > 188.05
2.30e6
0.26
0.39
MRM of 2 Channels ES+ 337.15 > 188.05
2.30e60.53
0.45
©2009 Waters Corporation | COMPANY CONFIDENTIAL 61
Final Chromatographic Method: Fentanyl
Time
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
%
0
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
%
0
Fentanyl_injsolv1_120209_001 MRM of 2 Channels ES+
342.05 > 187.95
1.42e70.93
Fentanyl_injsolv1_120209_001 MRM of 2 Channels ES+
337.15 > 188.05
1.42e7
0.93
Chromatographic Method
Atlantis HILIC SilicaLow pH
95 – 50% ACN in 2 min
75/25 ACN/MeOH with 0.2% HCOOH
N
N
O
CH3
Fentanylm.w. 336.47
Fentanyl
Fentanyl d5
©2009 Waters Corporation | COMPANY CONFIDENTIAL 62
OH
O
PCH3
O
Cyclohexyl methylphosphonic acid (CMPA)
CH3
O
O
PCH3
OH CH3
isobutyl hydrogen methylphosphonate (IBMPA)
O
O
POH
CH3
CH3
CH3
CH3CH3
Pinacolyl methylphosphonic acid (PMPA)
O
O
P
OHCH3
CH3
CH3
Isopropyl methylphosphonic acid (IMPA)
O
O
P
OHCH3
CH3
Ethyl methylphosphonic acid (EMPA)
Implementing the Approach:Example 2, Organophosphonic Acids
©2009 Waters Corporation | COMPANY CONFIDENTIAL 63
Stationary Phase Selectivity at Low pH:Organophosphonic Acids
Compounds1. PMPA2. CMPA 3. MMPA 4. IMPA 5. EMPA
500 ng/mL each
pH 3
Atlantis HILIC Silica yields greatest retention
BEH Amide and Atlantis HILIC Silica yield similar
selectivity2,3
0.00 1.00 2.00 3.00 4.00 5.00 6.00
0.00 1.00 2.00 3.00 4.00 5.00 6.00
0.00 1.00 2.00 3.00 4.00 5.00 6.00
1: SIR of 5 Channels ES-
TIC
3.32e6
1: SIR of 5 Channels ES-
TIC
3.32e6
1: SIR of 5 Channels ES-
TIC
3.32e6
1
2,3
45
1
4,5
1
2,3
4 5
BEH Amide
BEH HILIC
Atlantis HILIC Silica
Minutes
©2009 Waters Corporation | COMPANY CONFIDENTIAL 64
Compounds1. PMPA2. CMPA 3. MMPA 4. IMPA 5. EMPA
500 ng/mL each
BEH Amide
Higher sensitivity at pH 9
Similar selectivity independent of pH
Mobile Phase pH Selectivity:Organophosphonic Acids
0.00 1.00 2.00 3.00 4.00 5.00 6.00
0.00 1.00 2.00 3.00 4.00 5.00 6.00
1: SIR of 5 Channels ES-
TIC
3.32e6
1: SIR of 5 Channels ES-
TIC
3.32e6
1
2,3
45
1 2,3
45
pH 3
pH 9
Minutes
©2009 Waters Corporation | COMPANY CONFIDENTIAL 65
Compounds1. PMPA2. CMPA 3. MMPA 4. IMPA 5. EMPA
500 ng/mL each
pH 9
Greater Resolution for BEH Amide
No resolution between peaks 2 and 3
Further optimization needed
Stationary Phase Selectivity at High pH:Organophosphonic Acids
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
1: SIR of 5 Channels ES-
TIC
3.32e6
1 2,3
45
BEH Amide
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
1: SIR of 5 Channels ES-
TIC
3.32e6
1
2,3
45
BEH HILIC
©2009 Waters Corporation | COMPANY CONFIDENTIAL 66
Method Optimization Steps
1• Adjust gradient slope
2• Adjust column temperature
3• Adjust column length and flow rate
4
• Isocratic mode instead of gradient
• 95:5 ACN:H2O with 10 mM buffer or 0.2% additive
5
• Replace a portion of the water in the mobile phase with a less polar solvent [MeOH, EtOH or IPA]
Evaluate result after each step. Stop after criteria for success has been met
Consider injection solvent (sample diluent) if poor peak shape/resolution
©2009 Waters Corporation | COMPANY CONFIDENTIAL 67
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
12,3
45
99.9% to 0.1% B in 5 min
SIR of 5 Channels ES-TIC
3.32e6
2,3
4
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
1SIR of 5 Channels ES-
TIC4.18e6
99.9% to 50% B in 5 min
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
SIR of 5 Channels ES-TIC
5.12e6
12
3
45
99.9% to 90% B in 5 min
Compounds1. PMPA2. CMPA 3. MMPA 4. IMPA 5. EMPA
500 ng/mL each
BEH Amide, pH 9
Shallower gradient slope results in
improved resolution
Optimization Step 1:Adjust Gradient Slope
©2009 Waters Corporation | COMPANY CONFIDENTIAL 68
SIR of 5 Channels ES-TIC
5.08e6
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
21
3
4
5
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
21
34
5
30 °C
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
SIR of 5 Channels ES-TIC
5.12e6
12
3
45
SIR of 5 Channels ES-TIC
5.01e6
50 °C
65 °C
Compounds1. PMPA2. CMPA 3. MMPA 4. IMPA 5. EMPA
500 ng/mL each
BEH Amide, pH 9Shallow gradient
Increased temperature results in improved resolution
Optimization Step 2:Column Temperature
©2009 Waters Corporation | COMPANY CONFIDENTIAL 69Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00
2
1
34
5
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
21
3
4
5
SIR of 5 Channels ES-TIC
2.1 x 50 mm
SIR of 5 Channels ES-TIC
2.1 x 100 mmCompounds1. PMPA2. CMPA 3. MMPA 4. IMPA 5. EMPA
500 ng/mL each
BEH Amide, pH 9Shallow gradient, 65 oC
100 mm column results in improved resolution
50 mm column results in shorter run time
Select result that meets method criteria
Optimization Step 3:Column Length
©2009 Waters Corporation | COMPANY CONFIDENTIAL 70
0.00 2.00 4.00 6.00 8.00 10.00 12.00
2
1
3
4
5
Compounds1. PMPA2. CMPA 3. MMPA 4. IMPA 5. EMPA
500 ng/mL each
Final Method:Organophosphonic Acids
©2009 Waters Corporation | COMPANY CONFIDENTIAL 71
Rapid HILIC Method Development
Screening approach Time c
Column conditioning* 30 minutes
3 Columns, 2 pH’s screening 30 minutes
Optimization
Column conditioning [temp. equilibration] 30 minutes
Gradient slope and temperature 30 minutes
Total method development time 2 Hours
*equilibration and 2 blank injections at each pH
©2009 Waters Corporation | COMPANY CONFIDENTIAL 72
Conclusions
For HILIC retention and selectivity:
— ACN is used the primary [weak] solvent in HILIC
— Water, methanol, ethanol or isopropanol are strong [elution] solvents
— Stationary phase charge and bonded phase can impact retention and selectivity
— Analytes in their charged form exhibit greater retention [acids at high pH, bases at
low pH]
Practical considerations:
— At least 10 mM buffer or 0.2% additive is recommended in mobile phase A and B
— Sample diluent should contain at least 75% acetonitrile for solubility and peak shape
— Weak needle wash must be in a high organic solution [90 – 95% ACN]
A systematic screening protocol was described to effectively and efficiently
develop HILIC methods
— 3 column selectivities, 2 mobile phase pH’s
— ACQUITY UPLC system used for rapid, automated method development
— Total method development time of 2 hours