©2007 Waters Corporation
Waters AAPS 2007 Seminars
November 12-13, 2007
©2007 Waters Corporation 2
TodayToday’’s Schedules Schedule
©2007 Waters Corporation 3
TomorrowTomorrow’’s Schedules Schedule
©2007 Waters Corporation
An Introduction to UPLCAn Introduction to UPLC®® Technology: Technology: Improve Productivity and Data Quality Improve Productivity and Data Quality
Doug McCabeDoug McCabe
©2007 Waters Corporation 5
AgendaAgenda
Introduction: What is UPLC® Technology?
Migrating an HPLC Method to a UPLC® Method
Efficient UPLC® Method Development and Validation
Conclusion
©2007 Waters Corporation 6
UPLCUPLC®® Technology & The Technology & The Fundamental Resolution EquationFundamental Resolution Equation
))((1k
k14N
Rs+
−=
αα
•In UPLC® systems, increasing N (efficiency) is the primary focus•Selectivity and retentivity are the same as in HPLC•Resolution, Rs, is proportional to the square root of N
If N ↑ 3x, Rs ↑ 1.7x
NRs∝
Physical Chemical
©2007 Waters Corporation 7
Improving Resolution with Smaller ParticlesImproving Resolution with Smaller ParticlesConstant Column LengthConstant Column Length
Efficiency (N), is inversely proportional to Particle Size, dp
Rs ↑ 1.7X N ↑ 3X,dp ↓ 3X,(e.g., 5 μm to 1.7 μm) (i.e., Rs α √N)
∝
©2007 Waters Corporation 8
0
5
10
15
20
25
30
0 1 2 3 4 5 6 7 8 9 10 11 12
Linear Velocity [mm/s]
HE
TP
(µ
m)
5 µm SunFire™ C18
3.5 µm SunFire™ C18
1.8 µm ACQUITY UPLC® HSS T3
1.7 µm ACQUITY UPLC® BEH C18
UPLCUPLC®® Particles, Particles, van Deemter Curves & van Deemter Curves & Flow RatesFlow Rates
8.407.707.006.305.604.904.203.502.802.101.400.704.6 mm ID
1.801.651.501.351.201.050.900.750.600.450.300.152.1 mm ID
0.420.390.350.320.280.250.210.180.140.110.070.041.0 mm ID
Flow rate (mL/min)
©2007 Waters Corporation 9
Resolution (and Speed)Resolution (and Speed)Constant Column LengthConstant Column Length
Plates, Flow Rate and Particle Size
10000900080007000600050004000300020001000
1.0 2.0 3.0
Flow Rate{mL/min}
N
1200011000
Smaller Particle
Larger Particle
Smaller Particle Size*Increased N,*Higher, optimal u*Increased pressure
Optimal
Optimal flow rate is inversely proportional to dp
Isocratic analysis time is inversely proportional to F
Rs
Rs ↑ 1.7X, N ↑ 3X, dp ↓ 3X, T ↓ 3X
dp1
Fopt ∝
(e.g., 5 μm to 1.7 μm) (Rs α √N)
©2007 Waters Corporation 10
Efficiency, N is inversely proportional to the square of Peak Width, W
Peak height is inversely proportional to Peak Width
Peak Width and SensitivityPeak Width and SensitivityConstant Column LengthConstant Column Length
2w1
N∝
w1
Height ∝
Rs ↑ 1.7X, N ↑ 3X, dp ↓ 3X, T ↓ 3X
Sensitivity ↑ 1.7X
(e.g., 5 μm to 1.7 μm) (Rs α √N)
©2007 Waters Corporation 11
BackpressureBackpressureConstant Column LengthConstant Column Length
Backpressure is proportional to Flow Rate, FR, andinversely proportional to Particle Size squared
Optimal Flow Rate is inversely proportional to Particle Size (further to the right on van Deemter curve)
2dp1
FRΔP ×∝
dp1
FRopt∝
P ↑ 27X (~1/dp3)
dp ↓ 3X,(e.g., 5 μm to 1.7 μm)
©2007 Waters Corporation 12
Constant Column LengthConstant Column LengthFlow Rate Proportional to Particle SizeFlow Rate Proportional to Particle Size
AU
0.000
0.010
0.020
0.030
0.040
0.050
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 15.00
4.8 µm, 0.2 mL/min, 354 psi
AU
0.000
0.010
0.020
0.030
0.040
0.050
Minutes
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Theory1.7X Resolution
3X Faster1.7X Sensitivity25X Pressure
Reality1.5X Resolution
2.6X Faster1.4X Sensitivity22X Pressure
1.7 µm, 0.6 mL/min, 7656 psi
2.1 x 50 mm columns Too Much Backpressure!
©2007 Waters Corporation 13
Column Length to Particle Size RatioColumn Length to Particle Size RatioIndicates Maximum Resolution CapabilityIndicates Maximum Resolution Capability
100mm1.7μm
μm
300mm10μm = 30,000
30,000150mm =5μm
100mm =3 33,300
50mm1.7μm
= 29,410
= 58,820
1970’s ~ 30 min.
1990’s ~ 10 min.
2004 ~1 - 2 min.
1980’s ~ 15 min.
L/dp RATIO Typical Run Times
2x Max. Resolution Capability
150mm1.7μm
= 88,230 3x Max. Resolution Capability
30 mm
50 mm
100 mm
150 mm
17,650
29,500
58,820
88,235
L/dp
2.5 μm
3.0 μm
3.5 μm
5.0 μm
8,000
6,667
5,714
4,000
L/dp
IS® Columns(20 mm Length)
dp
Length (L)
The SAME L/dp for 2 columns will produce the SAME Resolution.The difference: shorter columns will produce the separations faster.
©2007 Waters Corporation 14
Scaling HPLC to UPLCScaling HPLC to UPLC®® SeparationsSeparationsConstant L/Constant L/dpdp = Equivalent Resolution= Equivalent Resolution
HP
LC
Sep
ara
tio
ns
UP
LC
®
Sep
ara
tio
n
2.5 µm – 75 mmInjection = 2.5 µL
Flow rate = 0.5 mL/minRs (2,3) = 2.34
5 µm – 150 mmInjection = 5.0 µL
Flow rate = 0.2 mL/minRs (2,3) = 2.28
3.5 µm – 100 mmInjection = 3.3 µL
Flow rate = 0.3 mL/minRs (2,3) = 2.32
1.7 µm – 50 mmInjection = 1.7 µL
Flow rate = 0.6 mL/minRs (2,3) = 2.29
©2007 Waters Corporation 15
Speed Increases Speed Increases Constant L/dpConstant L/dp
Efficiency, N, is directly proportional to column length, L, and
inversely proportional to particle size, dp:
For same N and, therefore, same Rs
dpL
N∝
N = 1X, L ↓ 3X,dp ↓ 3X, Rs = 1X,
F ↑ 3X, T ↓ 9X Efficiency & Resolution
Remain Unchanged
(e.g., 5 μm to 1.7 μm) (e.g., 150 mm to 50 mm)
(i.e., F increases 3X,L decreases 3X)
©2007 Waters Corporation 16
Assuming same efficiency, Peak Height is inversely proportional to column length, L:
For same efficiency, column length, L is decreased proportionally to particle size, dp (constant L/dp)
Sensitivity IncreasesSensitivity IncreasesConstant L/dpConstant L/dp
L1
Height =
N = 1X, L ↓ 3X, dp ↓ 3X, Rs = 1X,
Sensitivity ↑ 3X T ↓ 9X, Efficiency & Resolution
Remain Unchanged
(e.g., 5 μm to 1.7 μm) (e.g., 150 mm to 50 mm)
(Increased optimal flow rate & shorter column)
(Peak height increases as peak width and column length decreases)
©2007 Waters Corporation 17
Backpressure (P) is proportional to Column Length, L:
For constant L/dp, Backpressure is inversely proportional to the square of Particle Size, dp:
Backpressure Increases Backpressure Increases Constant L/dpConstant L/dp
LP∝
2dp1
P∝
P ↑ 9X L ↓ 3X, dp ↓ 3X, (e.g., 5 μm to 1.7 μm) (e.g., 150 mm to 50 mm)
©2007 Waters Corporation 18
AU
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0.04
0.06
Minutes
0.00 5.00 10.00 15.00 20.00 25.00 30.00
4.8 µm, 100 mm, 0.2 mL/min
Length Proportional to Particle SizeLength Proportional to Particle SizeSimilar L/dpSimilar L/dp
AU
0.00
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0.04
0.06
Minutes
0.00 1.00 2.00 3.00 4.00
RealitySame Resolution
8X Faster2.5X Sensitivity
11X Back Pressure
1.7 µm, 30 mm, 0.6 mL/min
TheorySame Resolution
9X Faster3X Sensitivity
9X Back Pressure
2.1 mm ID columnsManageable Backpressure
Increase
©2007 Waters Corporation 19
UPLCUPLC®® Technology & Gradient Technology & Gradient Peak Capacity (Resolution)Peak Capacity (Resolution)
-0.005
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50
tg
w ww w
w
wt
1Pc g+=
Gradient Duration
Peak Width
w ↓, Pc ↑
Peak capacity is a measure of the separation power of a gradient on a particular column
Pc = # Peaks separated per gradient duration time
©2007 Waters Corporation 20
UPLCUPLC®® Technology & Gradient Technology & Gradient Peak Capacity (Resolution)Peak Capacity (Resolution)
Peak capacity is affected by:— Gradient duration (tg)
— Flow rates (F)
— Column length (L)
— Particle size (dp)
Remember, the flow rate, column length and particle size all influence the plate count (N in isocratic separations)
By optimizing these parameters, peak capacities can be maximized
Influences peak width
©2007 Waters Corporation 21
Dependence of Peak Capacity on Dependence of Peak Capacity on Operating ConditionsOperating Conditions
1tt
ΔcB
ΔcB4
tL
Dd
cLt
Dbda
L
1P
g
0
0M
2p0
Mp
+⋅⋅
⋅⋅
⋅⋅+⋅⋅+⋅+=
We can now generate a 3-dimensional plot to examine how the gradient time and flow rate effect the peak capacity of a separation.
wt
1Pc g+=Start with the simple equation for peak capacity
Make a few substitutions
Neue, U. D., Mazzeo, J. R. J. Sep. Sci. 2001, 24, 921-929.Cheng, Y-F., Lu, Z., Neue, U. Rapid Commun. Mass Spectrom. 2001, 15, 141-151.
©2007 Waters Corporation 22
GradientDuration
(min)
FlowRate
(mL/min)
Pressure(psi)
Max PeakCapacity
1 0.352 3621 634 0.124 1280 10016 0.088 905 13632 0.062 640 151
Effect of Particle Size on Peak Effect of Particle Size on Peak Capacity for UPLCCapacity for UPLC®® SeparationsSeparations
Gradient Duration
(min)
Flow Rate
(mL/min)
Pressure(psi)
Max Peak Capacity
1 0.249 10852 1084 0.249 10852 17216 0.124 5426 21632 0.088 3837 231
Gradient Duration
(min)
Flow Rate
(mL/min)
Pressure(psi)
Max Peak Capacity
1 0.35 1774 464 0.124 627 7616 0.062 314 10732 0.044 222 121
ACQUITY UPLC® Columns can provide better Peak Capacity in 1 minute, than a 5 μm column in 16 minutes!!
1.0 x 50 mm Columns Pmax = ~11,000 psi
0.00
30.
004
0.00
50.
008
0.01
10.
016
0.02
20.
031
0.04
40.
062
0.08
80.
124
0.17
60.
249
0.35
20.
498
0.70
40.
995
1.40
71.
990
12
48163264
0
50
100
150
200
250
0.00
30.
004
0.00
50.
008
0.01
10.
016
0.02
20.
031
0.04
40.
062
0.08
80.
124
0.17
60.
249
12
48
1632
64
0
50
100
150
200
250
0.00
30.
004
0.00
50.
008
0.01
10.
016
0.02
20.
031
0.04
40.
062
0.08
80.
124
0.17
60.
249
0.35
20.
498
0.70
40.
995
12
4816
3264
0
50
100
150
200
250
Peak C
ap
aci
ty
Flow Rate (mL/min)
1.7 μm 3.5 μm 5 μm
108
100 107
Gra
dien
t D
ura
tion
(min
)
©2007 Waters Corporation 23
High Resolution Peptide Mapping:High Resolution Peptide Mapping:Influence of Particle SizeInfluence of Particle Size
AU
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UPLC® Gradient1.7 µm
Peaks = 168Pc = 360
2.5X Increase
HPLC Gradient5 µm
Peaks = 70Pc = 143
More information in the same amount of time
©2007 Waters Corporation 24
Summary:Summary:UPLC® Technology: What is it?
UPLC® Technology is based on chromatographic theory (not marketing) and utilizes:— Small, pressure-tolerant particles that are efficiently
packed into short (fast) or long (high resolution) columns
— An LC system that can operate at the optimal linear velocity (and resulting pressure) for these particles and possesses minimal system volume and fast, responsive detectors that do not negatively affect efficiency
UPLC® Technology provides information in less time and, hence, at a lower cost
©2007 Waters Corporation 25
AgendaAgenda
Introduction: What is UPLC® Technology?
Migrating an HPLC Method to a UPLC® Method
Efficient UPLC® Method Development and Validation
Conclusion
©2007 Waters Corporation 26
Example: Migrating an HPLC Method to a Example: Migrating an HPLC Method to a UPLCUPLC®® MethodMethod
AU
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0.30
0.40
Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
UPLC® MethodPc = 85
HPLC MethodPc = 94
AU
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©2007 Waters Corporation 27
Method Migration/Conversion: Method Migration/Conversion: HPLC to UPLCHPLC to UPLC®® TechnologyTechnology
Why Convert HPLC Methods to UPLC® Technology?— Acquire results in less time and/or with more resolution
o More information - faster
o More robust methods – greater confidence
o Better situational response time (stat samples faster, research decisions with more information, process monitoring, product release)
o More samples analyzed per system, per scientist
©2007 Waters Corporation 28
Channel: W2996 238.0nm-1.2; Processed Channel: W2996 PDA 238.0 nm at 1.2; Injection: 3; Date Acquired: 10/5/2006 10:12:29 AM EDT; Result Id: 1318; Processing Method: Simvastatin BEH 4_6 x 250
9.28
1
AU
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USP USP HPLCHPLC Separation of Separation of SimvastatinSimvastatin
O
OO
OOH
CH3 CH3 CH3
CH3
CH3H
Plates = 12,112USP Method Requirements
k’ > 3.0N > 4,500Tf < 2.0
HPLC:RT = 9.28 min
k’ = 4.1N = 12,112
Tf = 1.1
©2007 Waters Corporation 29
UPLCUPLC®® Method Conversion Method Conversion ChoicesChoices
Sim
vast
atin
- 1.
412
AU
0.00
0.07
0.14
0.21
Minutes0.00 0.50 1.00 1.50
Sim
vast
atin
- 1.
921
AU
0.00
0.07
0.14
0.21
Minutes0.00 0.50 1.00 1.50 2.00 2.50
Sim
vast
atin
- 0.
234
AU
0.00
0.03
0.06
0.09
Minutes0.00 0.50 1.00 1.50
Maximum EfficiencyMaximum Efficiency Equal EfficiencyEqual Efficiency Fastest AnalysisFastest Analysis
Efficiency = 12874Efficiency = 17685 Efficiency = 977
UPLC®:RT = 1.41 min
k’ = 4.9N = 12,874
Tf = 1.1
HPLC:RT = 9.28 min
k’ = 4.1N = 12,112
Tf = 1.1
USP Req.k’ > 3.0
N > 4,500Tf < 2.0
©2007 Waters Corporation 30
Critical Caveat of Method Critical Caveat of Method ConversionConversion
The new method will be different from the original method—Operating conditions, e.g., flow rate
—Run time
—Appearance
—Of course, the objective was an improved method
The new method must preserve critical parameters—Complete resolution of all relevant analytes
—Peak homogeneity/purity
—Certainty of peak identification
—Quantitative accuracy and precision
©2007 Waters Corporation 31
Method Conversion ProcessMethod Conversion ProcessSteps for SuccessSteps for Success
1. Gather information about existing method and results
2. Select new or target column— Chemistry
— Dimensions
3. Compare instruments
4. Calculate method conversion conditions
5. Evaluate results of transfer
6. Optimize as required
©2007 Waters Corporation 32
1. Gather Required Information 1. Gather Required Information Original Method & ResultsOriginal Method & Results
Column — Chemistry (ligand, brand,
particle size)— Dimensions
Conditions— Mobile phase— Flow rate— Gradient profile, including
regeneration and reequilibration
— TemperatureSample— Diluent— Concentration— Molecular weight(s)— Injection volume
Chromatogram— Number of peaks
— Retention
— Resolution (critical pairs)
Quantitation— Limit of detection
— Limit of quantitation
— Linear dynamic range
— Accuracy
— Precision
©2007 Waters Corporation 33
2. Select Target Column 2. Select Target Column ACQUITY UPLCACQUITY UPLC®® Column ChemistriesColumn Chemistries
UPLC® Column Chemistries— ACQUITY UPLC® BEH C18— ACQUITY UPLC® BEH Shield RP18— ACQUITY UPLC® BEH C8 — ACQUITY UPLC® BEH Phenyl— ACQUITY UPLC® BEH HILIC— ACQUITY UPLC® HSS T3— ACQUITY UPLC® HSS C18— ACQUITY UPLC® HSS C18 SB
Check Column Selection Chart for closest match to original— Chromatographic test at pH 7— Provides an assessment of a column’s hydrophobicity and
base/neutral selectivity— Can be used to select “equivalent” columns for methods transfer
©2007 Waters Corporation 34
Waters ReversedWaters Reversed--Phase Column Phase Column Selectivity ChartSelectivity Chart
YMC-Pack™ ODS-A™
YMC J'sphere™ ODS–H80
ACQUITY UPLC® BEHXBridge™
C18
(ln [k] acenaphthene)102007
SunFire ™ C18
YMC-Pack™ PolymerC18™
Hypersil® CPS Cyano
YMC-Pack™ CN
Waters Spherisorb® S5 P
Hypersil® BDS PhenylNova-Pak® Phenyl
YMC-Pack™Phenyl
Hypersil® PhenylInertsil® Ph-3
YMC-Pack™ Pro C4™
YMCbasic™
Symmetry® C8YMC-Pack™ Pro C8™
Nova-Pak®C8
XTerra® MS C18 Symmetry® C18
YMC-Pack™Pro C18™
Inertsil® ODS-3
Nova-Pak®C18
YMC J'sphere™ODS–L80 Nucleosil® C18
Waters Spherisorb® ODS2
Waters Spherisorb® ODS1Resolve® C18
µBondapak™ C18
YMC-Pack™ ODS–AQ™
YMC J'sphere™ ODS–M80
Inertsil® CN-3
Waters Spherisorb® S5CN
Nova-Pak® CN HP
SymmetryShield™ RP8
SymmetryShield™ RP18
XTerra® RP8
XTerra® RP18
-0.6
-0.3
0
0.3
0.6
0.9
1.2
1.5
1.8
2.1
2.4
2.7
3
3.3
3.6
-1.5 -0.5 0.5 1.5 2.5 3.5
(ln [α
] am
itrip
tylin
e/ac
enap
hthe
ne)
XTerra® MS C8 Luna ® C18 (2)
XTerra ®Phenyl Luna ™
Phenyl Hexyl
ChromolithTM
RP-18Atlantis® dC18 Zorbax® XDB C18
ACT Ace® C18
Zorbax® SB C18
SunFire ™ C8
Luna®C8 (2)
ACQUITY UPLC® BEHXBridge™Shield RP18
ACQUITY UPLC® BEH XBridge™
C8
ACQUITY UPLC® BEH XBridge™
Phenyl
Atlantis® T3
ACQUITY UPLC® HSS T3
ACQUITY UPLC® HSS C18
ACQUITY UPLC® HSS C18 SB
©2007 Waters Corporation 35
3. Required Information 3. Required Information Original InstrumentOriginal Instrument
Mode of gradient generation—Single pump with gradient proportioning valve (GPV)
—Dual pump
—Brand and model number
System volume (dwell volume or delay volume)—Value and method used to measure
Injection mechanism
Mode of detection
©2007 Waters Corporation 36
3. System Volumes of Pumping 3. System Volumes of Pumping SystemsSystems
DetectorInjector Column
Pump 1
Smaller System Volume = Smaller Dwell volume
Pump 2
Mixer
Multi-Pump (High Pressure)
Gradient Proportioning Valve
DetectorInjectorAB
CD
Column Solventdelivery
Larger System Volume = Larger Dwell volume
Single Pump (Low Pressure)
Volume: From where mobile phase is mixed, to
where it enters the column.
©2007 Waters Corporation 37
Compensating for System Volume Compensating for System Volume DifferencesDifferences
Compare system volumes— This volume should be converted to column volumes for the
best comparison
If target system gives smaller isocratic segment— ADD an initial hold to the gradient table to give the identical
hold
If target system gives larger isocratic segment— No exact compensation is possible
— Chromatographic effect of extra isocratic hold usually small
ACQUITY UPLC® Columns Calculator handles this compensation
©2007 Waters Corporation 38
3. Instrument Comparison3. Instrument ComparisonInjectionInjection
We assume—The specified volume of sample is delivered to the
column
—The sample composition is not altered
—There is no carryover
System differences affect—Volume of sample required
—Absolute amount injected
—Sample carryover
©2007 Waters Corporation 39
ACQUITY UPLCACQUITY UPLC®® SystemSystemSample ManagerSample Manager
Minimize sample dispersion during injection
Reduce cycle time
Preserve—Accuracy
—Precision
—Low carryover
—Sample format flexibility
Dual wash system with a strong wash followed by a weak wash
©2007 Waters Corporation 40
3. Instrument Comparison 3. Instrument Comparison DetectionDetection
We assume—Response is specific
—Response is linear with concentration
—Detector does not alter peak shape
System differences affect—Specificity
—Limit of detection (LOD)
—Limit of quantitation (LOQ)
—Linear dynamic range
—Band-broadening
©2007 Waters Corporation 41
Detection with UPLCDetection with UPLC®® SeparationsSeparationsUV, ELSD & FLRUV, ELSD & FLR
Only Waters ACQUITY UPLC® TUV or PDA—Minimize band-broadening
—Provide adequate sampling rate (S/N)
—Accurate wavelength
UPLC® ELSD and FLR detectors also available
Sensitivity and dynamic range are affected by reduced peak volume and by improved resolution
©2007 Waters Corporation 42
Data Acquisition RatesData Acquisition RatesImpact on UV DataImpact on UV Data
Minutes0.50 0.52 0.54 0.56 0.58 0.60 0.62 0.64 0.66 0.68 0.70 0.72 0.74
1 pt/s
2 pt/s
5 pt/s
40 pt/s
20 pt/s
10 pt/s
How many Data Points is
enough?
Simple Rule:15-20 Points per
Peak
The RATE the points are collected is
determined by how wide the peak is in
TIME at the baseline.
If a peak is only 1 second wide, then you need to collect
20 points in 1 second (20 Hz)
Peaks are ~ 1-2 seconds wide
©2007 Waters Corporation 43
Detection with UPLCDetection with UPLC®® Separations Separations MS and MS/MSMS and MS/MS
Waters MS systems designed for UPLC®
separations—Z-spray source minimizes band-broadening
—Quattro Premier XE, LCT Premier XE and Q-Tof Premier
—SQD & TQD designed specifically for ACQUITY UPLC®
system and separations
—ZQ single quadrupole data acquisition is compatible with UPLC® separations
Other Waters MS detectors can be operated with compatible sampling rates
©2007 Waters Corporation 44
Ready to Migrate: Target Conditions Ready to Migrate: Target Conditions Mobile Phase and Injection ParametersMobile Phase and Injection Parameters
Use exactly the same mobile phase—Alter only after evaluating transfer if optimization is
required
Use exactly the same sample—Same concentration
—Same diluents
ACQUITY UPLC® System needle wash— Use final gradient conditions as strong needle wash
(200 µL)
— Use initial gradient conditions as weak needle wash (600 µL)
©2007 Waters Corporation 45
AU
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4. Example: Original HPLC Method:4. Example: Original HPLC Method:Caffeic Acid Derivatives of Echinacea Purpurea
Pc = 94
1 23
4
5
Name Retention Time USP Resolution1. Caftaric acid 5.712. Chlorogenic acid 7.07 4.203. Cynarin 13.96 21.194. Echinacoside 16.54 10.165. Cichoic acid 20.32 17.14
©2007 Waters Corporation 46
Enter Existing HPLC Enter Existing HPLC Gradient Conditions Gradient Conditions
1. Enter column, system & analyte information
2. Enter HPLC gradient Time & %B
3. Select Pmax for UPLC®
separation
4. Press Calculate
HPLC results given here
Calculated for you
©2007 Waters Corporation 47
Five UPLCFive UPLC®® Gradient Choices GivenGradient Choices Given
Five Gradient Choices:
GeometricallyScaled
1. HPLC Linear Velocity2. UPLC® Linear Velocity
3. User Definedor
Optimally Scaled4. Maximum Peak
Capacity5. Shortest Analysis
Time
Select View for Detailed Gradient Profiles
©2007 Waters Corporation 48
UPLCUPLC®® Method Gradient Profiles Method Gradient Profiles ChoicesChoices
Original HPLC Gradient Method
Geometrically ScaledHPLC Linear Velocity
Geometrically ScaledUser Defined Flow Rate
Geometrically ScaledUPLC® Linear Velocity
Maximum Peak Capacity Equivalent
Analysis Time
Equivalent Peak Capacity Shortest
Analysis Time
©2007 Waters Corporation 49
Calculator Choice 1 (default):Calculator Choice 1 (default):Geometrically Scaled Gradient at HPLC Linear Velocity
Geometrically Scaled UPLC® Gradient at HPLC Linear Velocity:Identical Column Volumes per Time Segment
Flow rate scaled for column ONLY
Original HPLC GradientUPLC® Gradient:
HPLC Linear Velocity
©2007 Waters Corporation 50
AU
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Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00
Calculator Choice 1 (default):Calculator Choice 1 (default):Geometrically Scaled Gradient at HPLC Linear Velocity
HPLC Method
UPLC® MethodChoice 1
Pc = 94
Pc = 99
Similar Resolution35.00
NameRetention Time
USP Resolution
caftaric acid 5.71chlorogenic acid 7.07 4.20cynarin 13.96 21.19echinacoside 16.54 10.16cichoic acid 20.32 17.14
NameRetention Time
USP Resolution
caftaric acid 1.99chlorogenic acid 2.38 3.97cynarin 4.85 25.00echinacoside 5.93 15.09cichoric acid 7.11 19.62
©2007 Waters Corporation 51
Calculator Choice 2:Calculator Choice 2:Geometrically Scaled Gradient at UPLC® Linear Velocity
Geometrically Scaled UPLC® Gradient at UPLC® Linear Velocity:
Identical column volumes per Time SegmentFlow rate scaled for column AND particle size
Original HPLC GradientUPLC® Gradient:
UPLC® Linear Velocity
©2007 Waters Corporation 52
AU
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Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
Calculator Choice 2:Calculator Choice 2:Geometrically Scaled Gradient at UPLC® Linear Velocity
NameRetention Time
USP Resolution
caftaric acid 5.71chlorogenic acid 7.07 4.20cynarin 13.96 21.19echinacoside 16.54 10.16cichoic acid 20.32 17.14
HPLC Method
NameRetention Time
USP Resolution
caftaric acid 0.71chlorogenic acid 0.87 3.96cynarin 1.72 22.12echinacoside 2.06 11.40cichoric acid 2.44 14.28
UPLC® MethodChoice 2
Pc = 94
Pc = 85
Similar Resolution
AU
0.00
0.10
0.20
0.30
0.40
Minutes0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00
©2007 Waters Corporation 53
UPLCUPLC®® Method Gradient Profiles Method Gradient Profiles ChoicesChoices
AU
0.00
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0.20
0.30
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Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00
AU
0.00
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Minutes
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00
AU
0.00
0.10
0.20
0.30
0.40
Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
Minutes0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00
Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
Original HPLC Gradient Method
Geometrically ScaledHPLC Linear Velocity
Geometrically ScaledUPLC® Linear Velocity
Equivalent Peak Capacity Shortest
Analysis Time
Maximum Peak Capacity Equivalent
Analysis Time
Similar Selectivities Different Selectivities
©2007 Waters Corporation 54
SummarySummaryHPLC to UPLC® Method Conversion
Methods can be moved directly from HPLC to ACQUITY UPLC® technology for— Improved resolution
— Improved speed
— Improved detectability
Attention to detail leads to success
ACQUITY UPLC® Columns Calculator facilitates process
Converting to UPLC® technology/methodology increases profitability by lowering cost of sample analysis and various connected operating costs
©2007 Waters Corporation 55
AgendaAgenda
Introduction: What is UPLC® Technology?
Migrating an HPLC Method to a UPLC® Method
Efficient UPLC® Method Development and Validation
Summary
©2007 Waters Corporation 56
Pharmaceutical Application AreasPharmaceutical Application Areas
Forced Degradation Studies
Stability Indicating Studies
Impurity Profiling and Identification
Quantitative Bioanalysis
Batch Scale Up Studies
Batch Comparison
Raw Material Acceptance
Finished Product Release Testing
Cleaning Validation
Patent Protection
©2007 Waters Corporation 57
UPLC Methods Development Protocol2.1 x 50 mm, 1.7 µm, 0.5 mL/minpH 3/ acetonitrile TimeFlow ramp 5 minColumn conditioning (2 blank gradients) 11 minSample injection (2 replicates) 11 minpH 3/ methanolFlow ramp 5 minColumn conditioning (2 blank gradients) 11 minSample injection (2 replicates) 11 minColumn purge 6 minpH 10/ acetonitrileFlow ramp 5 minColumn conditioning (2 blank gradients) 11 minSample injection (2 replicates) 12 minpH 10/ methanolFlow ramp 5 minColumn conditioning (2 blank gradients) 11 minSample injection (2 replicates) 11 minColumn purge 6 min
120 minSCREENING TIME 2 Hours/ Hybrid column
x 3 columns
1 Hour/ Silica columnx 1 column
TOTAL SCREENING TIME 7 HOURS
UPLC® Screening: 6.1X faster than 5.0 µm HPLC Column
Why Develop Methods with UPLCWhy Develop Methods with UPLC®® Technology?Technology?Time Savings Versus 5 µm HPLC Column
EQUIVALENT HPLC Methods Development Protocol, 5 µm4.6 x 150 mm, 5 µm, 1.0 mL/minpH 3/ acetonitrile TimeFlow ramp 5 minColumn conditioning (2 blank gradients) 79.2 minSample injection (2 replicates) 79.2 minpH 3/ methanolFlow ramp 5 minColumn conditioning (2 blank gradients) 79.2 minSample injection (2 replicates) 79.2 minColumn purge 43.2 minpH 10/ acetonitrileFlow ramp 5 minColumn conditioning (2 blank gradients) 79.2 minSample injection (2 replicates) 79.2 minpH 10/ methanolFlow ramp 5 minColumn conditioning (2 blank gradients) 79.2 minSample injection (2 replicates) 79.2 minColumn purge 43.2 min
740 minSCREENING TIME 12.3 Hours/ Hybrid column
x 3 columns
6.15 Hours/ Silica columnx 1 column
TOTAL SCREENING TIME 43 HOURS
©2007 Waters Corporation 58
Automated Method Development and Automated Method Development and ValidationValidation
Automated Method Development— ACQUITY UPLC® Column
Manager, 4 column selection device
— ACQUITY UPLC® Binary Solvent Manager, solvent select valves
Automated Method Validation— Empower® 2 Method Validation
Manager (MVM) streamlines method validation process
©2007 Waters Corporation 59
Column Chemistry
Solvent pHα
Selectivity
Selectivity ToolsSelectivity Tools
αSelectivity
©2007 Waters Corporation 60
Effect of Mobile Phase pHEffect of Mobile Phase pH
Affects only analytes with ionizable functional groups— Amines
— Carboxylic acids
— Phenols
Some compounds contain one or more ionizable function
Strongest selectivity effects can be caused by pH changes
©2007 Waters Corporation 61
pH
0
5
10
15
20
25
30
35
40
0 2 4 6 8 10 12
Ret
entio
n Fa
ctor
(k)
Acid
Base
Neutral
Note: Retention of neutral analytes not affected by pH
Increasedbase retention
Increased acid retention
Silica pH Range
Hybrid Particle pH Range
Neue et. al. American Laboratory 1999 (22) 36-39.
ReversedReversed--Phase Retention Map:Phase Retention Map:The Importance of Mobile Phase pHThe Importance of Mobile Phase pH
BEH
HSS
©2007 Waters Corporation 62
Column ChemistryLigand & Base particle
Solvent pH
αSelectivity
Selectivity ToolsSelectivity Tools
αSelectivity
©2007 Waters Corporation 63
Waters UPLCWaters UPLC®® Particles OverviewParticles Overview
Ethylene Bridged Hybrid (BEH) Particles—Wide pH range (1-12)—Five chemistries—Seamless HPLC → UPLC® method migration – with same
selectivity as XBridgeTM HPLC columns—130Å and 300Å pore diameters
High Strength Silica (HSS) Particles—ONLY UPLC®-certified 100% silica particle—Three C18 chemistries —Developed specifically for UPLC® applications—Packed, tested and guaranteed compatibility with at
pressures up to 15,000 psi (1000 bar)
©2007 Waters Corporation 64
The The ChemistriesChemistries of UPLCof UPLC®®
Technology Technology
Sep2006
Jun 2007
Dec2007
Mar2004
Mar2005
Mar2005
Mar2005
Dec2005
LaunchDate
©2007 Waters Corporation 65
BEH C18
BEH C8
BEH Shield RP18
BEH Phenyl
1
43
5 8,9,137 10116
1412
1 43
52
7 810
9 116
141312
14352
87 109116 141312
2
1
4
35 8 710,11
9 61413122
HSS T31 435
7,810
9 116 1413122
Conditions : Columns: ACQUITY UPLC® BEH 2.1 x 100 mm, 1.7 µm
ACQUITY UPLC® HSS 2.1 x 100 mm, 1.8 µmMobile Phase A: H2OMobile Phase B: MeOHFlow Rate: 0.5 mL/min Isocratic: 28% MeOHInjection Volume: 5.0 µLSample Concentration: 10 µg/mLTemperature: 50 oCDetection: UV @ 254 nmSampling rate: 20 pts/secTime Constant: 0.1Instrument: ACQUITY UPLC®with ACQUITY UPLC® PDA
HSS C18102
9431
8765
14131211
Selectivity ChoicesSeparations of Separations of NitroaromaticsNitroaromatics
Compounds1. HMX2. RDX3. 1,3,5-TNB4. 1,3-DNB5. NB6. Tetryl7. TNT8. 2-Am-4,6-DNT9. 4-Am-2,6 DNT10. 2,4-DNT11. 2,6-DNT12. 2-NT13. 4-NT14. 3-NT
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00
1122
33 44 5,65,677
88 99 1010 1111
1212 1313 1414 HSS C18 SB
©2007 Waters Corporation 66
Different LigandsDifferent Ligands::Different SelectivitiesDifferent Selectivities
Changes in hydrophobicity— Longer alkyl chain will provide greater retention
Changes in silanol activity— Affect peak asymmetry and influences secondary interactions
Changes in hydrolytic stability— Longer column lifetimes with greater number of ligand
attachment points to the particle surface
Changes in ligand density— Influences sample loadability
©2007 Waters Corporation 67
Low pH Selectivity DifferencesLow pH Selectivity Differences35%ACN/65% 15.4 mM HCOONH35%ACN/65% 15.4 mM HCOONH44, pH 3.0, pH 3.0
Y-AXIS X-AXISMaterial k (Tol) α (Ami/Tol) ln k (Tol) ln α (Ami/Tol)
BEH C18 13.01 0.365 2.57 -1.01
BEH C8 8.27 0.469 2.11 -0.76
BEH Shield RP18 11.19 0.256 2.42 -1.36
BEH Phenyl 6.60 0.613 1.89 -0.49
BEH 300 C18 5.97 0.409 1.79 -0.89
HSS C18 20.45 0.299 3.02 -1.21
HSS T3 17.91 0.393 2.89 -0.93HSS C18 SB 9.23 0.842 2.22 -0.17
BEH C8
BEH Phenyl
Shield RP18
BEH C18
BEH 300 C18
HSS T3HSS C18
HSS C18 SB
1.0
1.5
2.0
2.5
3.0
3.5
-1.75 -1.25 -0.75 -0.25 0.25
ln α (ami/tol) pH 3.0
lnK
(to
luen
e)
©2007 Waters Corporation 68
ACQUITY UPLCACQUITY UPLC®® USP USP ““LL””Designation Designation –– 2007/20082007/2008
August 2007: ACQUITY UPLC® Columns meet USP requirements are now officially considered “L” columns
©2007 Waters Corporation 69
Column Chemistry
Solvent pH
αSelectivity
Selectivity ToolsSelectivity Tools
αSelectivity
©2007 Waters Corporation 70
Solvent PropertiesSolvent Properties
Methanol—Weaker eluent
—H-bonding solvent
Acetonitrile—Aprotic solvent
—Stronger eluent
—Lower viscosity
©2007 Waters Corporation 71
Automated Method Development and Validation Automated Method Development and Validation Example: Example: ParoxetineParoxetine and Related Compoundsand Related Compounds
Method Development— Use systematic screening protocol
— Paroxetine (API) concentration: 0.2 mg/mL in 50:50 MeOH:H2O
— Related compounds at 10% concentration of API for easy identification during scouting
Method Optimization — Related compounds at 0.1% concentration of API
Paroxetinem.w. 374.8
©2007 Waters Corporation 72
ParoxetineParoxetine Related CompoundsRelated Compounds
Paroxetine HCl
— (-)-trans-4R-(4'-fluorophenyl)-3S-((3',4‘methylenedioxyphenoxy)methyl)piperidine
Paroxetine related compound B
— (trans-4-phenyl-3-[(3,4-methylenedioxy)phenoxymethyl]-piperidine HCl
Paroxetine related compound D
— (cis)-paroxetine HCl
Paroxetine related compound F
— Trans (-)-1-methyl-3-[(1,3-benzodioxol-5-yloxy)methyl]-4-(4-fluorophenyl)piperidine
Paroxetine related compound G
— [(+/-)Trans-3-[(1,3-benzodioxol-5-yloxyl)methyl]-4-(4”-fluorophenyl-4’phenyl)piperidine HCl
©2007 Waters Corporation 73
2. Column Chemistry
3. Solvent 1. pHα
Selectivity
Systematic Screening:Systematic Screening:Combining Chemical FactorsCombining Chemical Factors
αSelectivity
©2007 Waters Corporation 74
Stationary Phase Selectivity:Stationary Phase Selectivity:ParoxetineParoxetine
CM, ESG
Methanol pH 3.0
Poor resolution of paroxetine and Related Compounds (RC)
Observation:
Investigate high pHAction:
AU
0.00
0.10
0.20
0.30
0.40
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
ACQUITY UPLC® BEH C18
AU
0.00
0.10
0.20
0.30
0.40
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
AU
0.00
0.10
0.20
0.30
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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
AU
0.00
0.10
0.20
0.30
0.40
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
ACQUITY UPLC®
BEH Shield RP18
ACQUITY UPLC® BEH Phenyl
ACQUITY UPLC® HSS T3
B
GD FPa
roxe
tine
B GD
FParo
xetin
e
BGD
FParo
xetin
e
B
GD
FParo
xetin
e
©2007 Waters Corporation 75
pH Selectivity:pH Selectivity:ParoxetineParoxetine
CM, ESGCM, ESG
Better retention and resolution of API from RC due to neutral charge state of analytes at alkaline pH
Observation:
Select pH 10 due to better separation
Compare stationary phase selectivity with pH 10 buffer
Actions:
AU
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
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
pH 3.0Methanol
AU
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
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
pH 10Methanol
BGD
F
Paro
xetin
e
B GD F
Paro
xetin
e
©2007 Waters Corporation 76
Stationary Phase Selectivity:Stationary Phase Selectivity:ParoxetineParoxetine
CM, ESG
Methanol pH 10.0
Any column may provide successful separation
Observation:
Select ACQUITY UPLC® BEH C18
Compare selectivity between organic modifiers
Actions:
AU
0.00
0.10
0.20
0.30
0.40
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
ACQUITY UPLC® BEH C18
AU
0.00
0.10
0.20
0.30
0.40
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
AU
0.00
0.10
0.20
0.30
0.40
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
ACQUITY UPLC® BEH Shield RP18
ACQUITY UPLC® BEH Phenyl
B GD F
Paro
xetin
e
B GD F
Paro
xetin
e
B GD F
Paro
xetin
e
©2007 Waters Corporation 77
Solvent Selectivity:Solvent Selectivity:ParoxetineParoxetine
ACQUITY UPLC® BEH C18
CM, ESG
Methanol is weaker elution solvent resulting in greater retention
Better resolution exhibited with acetonitrile as organic modifier
Observations:
Select acetonitrile as organic modifier
Optimize separation using appropriate concentration of RC
Actions:
AU
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
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
AcetonitrilepH 10.0
AU
0.00
0.05
0.10
0.15
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0.25
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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
MethanolpH 10.0
BG
D FPa
roxe
tine
BGD F
Paro
xetin
e
©2007 Waters Corporation 78
Related Compounds at Related Compounds at 0.1% Concentration of 0.1% Concentration of ParoxetineParoxetine
Inadequate resolution among paroxetine and related compounds B and D due to disparate levels of concentration
Observation:
Change gradient slopeAction:
AU
0.00
0.02
0.04
0.06
0.08
0.10
Minutes2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00
AU
-0.003
-0.002
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
Minutes2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00
Related Compounds at 10%
Related Compounds at 0.1%
D
G
F
B
Paro
xetin
e
DG
F
B
Paro
xetin
e
©2007 Waters Corporation 79
AU
-0.003
-0.002
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
Minutes2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00
AU
0.002
0.003
0.004
0.005
0.006
0.007
0.008
Minutes2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00
Method Optimization:Method Optimization:Gradient SlopeGradient Slope
ACQUITY UPLC® BEH C18
CM, ESG
5 Minute Gradient5%-90%
5 Minute Gradient20%-90%
Acetonitrile pH 10.030oC
Marginal improvement in separation of impurities from parent compound with shallow gradient slope
Observations:
Alter gradient endpoint to produce shallower slope
Action:
DG
FB
Paro
xetin
e
DG
FB
Paro
xetin
e
©2007 Waters Corporation 80
AU
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
Minutes2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00
AU
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
Minutes2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00
Method Optimization:Method Optimization:Gradient SlopeGradient Slope
CM, ESG
5 Minute Gradient20%-90%
5 Minute Gradient20%-65%
Acetonitrile pH 10.030oC
Resolution remains inadequate with shallow gradient slope
Observations:
Investigate column temperature
Action:
D
GFB
Paro
xetin
e
DG F
B
Paro
xetin
e
©2007 Waters Corporation 81
Method Optimization:Method Optimization:Column TemperatureColumn Temperature
CM, ESG
Acetonitrile pH 10.0
Higher temperature improves separation of RC from paroxetine
Peak shape improves as temperature increases
Observations:
Select 60 oC for best resolution and peak shape
Action:
AU
0.002
0.004
0.006
0.008
Minutes2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00
AU
0.002
0.004
0.006
0.008
Minutes2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00
AU
0.002
0.004
0.006
0.008
Minutes2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00
30 oC
45 oC
60 oCD G FB Pa
roxe
tine
D G FB Paro
xetin
e
D G FB
Paro
xetin
e
©2007 Waters Corporation 82
Final Method Final Method ParoxetineParoxetine and Related Compounds at 0.1%and Related Compounds at 0.1%
AU
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
0.009
0.010
Minutes2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00
D G FBPa
roxe
tine
Compound USP RsRelated compound BParoxetine 1.95Related compound D 3.07Related compound G 13.00Related compound F 6.74
ACQUITY UPLC® BEH C18,, 2.1 x 50 mm, 1.7 µmMobile phase A: 20 mM NH4HCOO3, pH 10Temperature: 60 oC5 Min Gradient: 20%-65% ACNFlow rate: 0.5 mL/minInjection Volume: 4 µLDetection: UV @ 295 nm
AU
0.00
0.10
0.20
0.30
0.40
0.50
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00
©2007 Waters Corporation 83
Why Validate?Why Validate?
Ensures that analytical methodology is accurate, reproducible and robust over the specific range that an analyte will be analyzed
Provides assurance of reliability
FDA Compliance
Good Science!!
"The process of providing documented evidence demonstrating that something (the method or procedure) does what it is intended to do; is suitable for its intended purpose."
©2007 Waters Corporation 84
Method ValidationMethod Validation
Waters cannot define that for you
You must define or follow your own corporate policies
However, there are some strategies colleagues are using today — Full Blown Validations (conservative approach)
— Cross validations
o SLAP technique– perform selectivity, linearity, area, and precision tests
Relate specifications to critical quality attributes— Propose acceptance criteria based on scientific rationale
— Relationships established from DOE and prior knowledge
Which Validation Steps need to be performed after a method has been converted to UPLC® technology?
Automation tools can be used to facilitate the process
©2007 Waters Corporation 85
New Method Validation Manager New Method Validation Manager Option For Empower 2Option For Empower 2
"The industry is very much in need of a workflow-based, configurable system that allows users to implement an organization’s method validation practices. An approach that seamlessly implements method validation requirements but is inherently flexible and manages the data, effectively standardizes an often tedious and time-consuming process“— James Morgado, Pfizer Global R&D
Method Validation Manager option automates the laborious method validation process within Empower 2 Software
Realize time savings of 50% – 80% in this formerly manual and error-prone iterative process
©2007 Waters Corporation 86
Data Acquisition& Processing
Time consuming, repetitive tasks consisting of Time consuming, repetitive tasks consisting of several sequential stepsseveral sequential stepsFaster and Easier Method ValidationFaster and Easier Method Validation
Corporate Method ValidationSOP
MethodValidationManager
Analytical Method Validation Process with Analytical Method Validation Process with Method Validation ManagerMethod Validation Manager
PrepareStandards & Samples
Data Management
Create SampleSequence
Calculation Statistical Results
Reports Compiled
©2007 Waters Corporation 87
Automated Method Validation Manager:Automated Method Validation Manager:ParoxetineParoxetine Validation ResultsValidation Results
pass0.00 – 2.57% RSDVariance Component < 5 %RSD Retention Time
(Buffer strength, Additive Conc., Column Temperature, Flow Rate, Injection Volume)
Method Robustness
Retention Time
pass0.06 – 1.64% RSDVariance Component < 2 %RSD Peak Area
(Buffer strength, Additive Conc., Column Temperature)
Method Robustness
Peak Area
pass0.05% of active at 0.2 mg/mL (s/n 2.2 – 6.23)
Impurities 0.1% of active at 0.2 mg/mLLOD of impurities
pass
pass
pass
Analyst 1.33% RSD
Instrument 7.72% RSD
Column 0.00% RSD
Variance Component < 10 %RSD Peak Area
(Analyst, Instrument, Column)
Intermediate Precision
pass97 – 102 %80 – 120 %Accuracy
pass
pass
0.9999
1.74% RSD
R2 > 0.995
Residuals < 2.0% RSD
Linearity
Pass/FailReported ValueAcceptance CriteriaParameter
©2007 Waters Corporation 88
Methods Development and Validation Timeline:Methods Development and Validation Timeline:Instrument Time
UPLC® Methods Development and Validation Timeline2.1 x 50 mm, 1.7 µm, 0.5 mL/min
Screening Time4 Columns, 2 Organics, 2 pH’s 7.0 hours
OptimizationGradient Slope and Temperature 1.7 hours
ValidationAccuracy, linearity, repeatability,Reproducibility, LOD/LOQ, Intermediate precision, robustness 21.1 hours
TOTAL TIME 29.8 HOURS
©2007 Waters Corporation 89
Method Validation ManagerMethod Validation ManagerBenefitsBenefits
Save time over entire process and less error prone— Data management is handled by Empower 2, not by user
— Automatic data checks performed at each step of the workflow
— Data approvals can be configured at each step of the workflow
— Calculations done in Empower 2
o No transfer to spreadsheets or other software
o No transcription error / No need to check data transfer
o No need to validate spreadsheet functions
o Multi-component analysis and batch processing of validation results
— Report templates can be used to standardize the report format
o Automatic report generation
o Ease of Review
©2007 Waters Corporation 90
Summary:Summary:Efficient UPLCEfficient UPLC®® Method Development and ValidationMethod Development and Validation
Achieve more resolution, faster by utilizing sub-2 µm UPLC®
columns at optimal linear velocities with full pressure capabilities up to 15,000 PSI
Principles of methods development remain the same
UPLC® column chemistries provide a broad range of selectivity to successfully develop methods efficiently
UPLC® Technology allows for faster methods development and validation
UPLC® Technology, Empower® 2 and Method Validation Manager software can significantly improve laboratory productivity and compliance
©2007 Waters Corporation 91
AgendaAgenda
Introduction: What is UPLC® Technology?
Migrating an HPLC Method to a UPLC® Method
Efficient UPLC® Method Development and Validation
Conclusion
©2007 Waters Corporation 92
ConclusionConclusion
UPLC® Technology provides more reliable information FASTER and at a LOWER cost per analysis
UPLC® Technology is NOT:— Just fast LC (speed WITHOUT resolution)
— 2.X µm HPLC particles packed into short (≤100 mm) HPLC column hardware that are run on an HPLC system under HPLC operating pressures (beware of these claims)
UPLC® Technology and Empower 2 software can improve productivity and compliance by streamlining method development and validation protocols
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©2007 Waters Corporation 94
Presentation Presentation pdfpdf files available on files available on www.waters.comwww.waters.com/slides/slides
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