finland uplc users meeting mai, 22th - waters corporation · ©2012 waters corporation 1 finland...
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©2012 Waters Corporation 1
Finland UPLC Users MeetingMai, 22th
Frederic ForiniWaters European Headquarters
©2012 Waters Corporation 2
What is the USP?What is the USP?� The United States Pharmacopeia (USP) is an independent, official public standards-setting authority – Prescription and OTC medicines– Healthcare products– Food ingredients – Dietary supplements
� USP standards recognized and used in >130 countries
� Head quarters and offices
©2012 Waters Corporation 3
What is the USPWhat is the USP--NF?NF?� The United States Pharmacopeia – National Foundry (USP-NF) is a book of pharmacopeial standards– Drugs substances & preparations monographs: USP– Dietary supplements & ingredients monographs: USP– Excipient monographs: NF– More than 4500 monographs
� The USP-NF is the official authority – FDA-enforceable standards– Enforcement of USP standards is the responsibility of FDA and
other government authorities in the U.S. and elsewhere– USP has no role in enforcement
The U.S. Federal Food, Drug, and Cosmetics Act designates the USP–NF as the official compendia for drugs marketed in the
United States
©2012 Waters Corporation 4
What is the What is the PharmacopeialPharmacopeial Forum (PF)Forum (PF)� USP standards are established and maintained through public participation1. Sponsors provide draft standards and supporting data2. USP scientific staff and volunteer experts review, test and forward
new/revised monograph to PF3. Monograph is refined and finalized through public review and
comment in PF
� PF is FREE bimonthly online journal where public review and comment takes place
©2012 Waters Corporation 5
Why are Such Changes to Why are Such Changes to Chromatography <621> Important?Chromatography <621> Important?� Question From CURRENT USP-NF Online FAQs1
– Q. How much can I modify a chromatographic procedure and still be in compliance? Can column length, internal diameter, mobile phase composition be modified?
– A. Chromatography <621> contains a list of allowed adjustments to chromatographic systems. However, the user should verify the suitability of the method under the new conditions by assessing the relevant analytical performance characteristics potentially affected by the change (see section System Suitability under Chromatography <621>).
1http://www.uspnf.com/uspnf/scienceFAQ.html#q4
©2012 Waters Corporation 6
Working with USP: Working with USP: ACQUITY UPLC Columns “L” ListingACQUITY UPLC Columns “L” Listing� Back in August 2007: ACQUITY UPLC® Columns included in USP LC columns “L” listings– Waters was instrumental in changing minimum particle size
©2012 Waters Corporation 7
Current Regulation Allowed Current Regulation Allowed AdjustmentsAdjustments� Chromatography <621> describes the adjustments allowed in the chromatography system when system suitability test fails
Allowed HPLC Adjustment
USP (Ref: General Chapter <621>) EP (Ref: General Chapter 2.2.46)
Column Length ±70% ±70%
Internal Diameter Can be adjusted if linear velocity is kept constant ±25%
Particle Size Reduction of 50%, no increase Reduction of 50%, no increase
Flow Rate±50% or more as long as the linear velocity is kept
contant ±50%
Column Temperature ±10C ±10% Max 60C
Injection Volume Change allowed as long as SST criteria are met May be decreased (if LOD and repeatability ok)
PH ±0.2 Units ±0.2 Units (±1% for neutral substances)
UV wavelength <±3 nm <±3 nm
Conc. Salts in Buffer ±10% ±10%Composition of mobile phase
Minor Components (<50%) ±30% or ±10% absolute whichever is smaller
Minor Components ±30% or ±2% absolute whichever is larger
©2012 Waters Corporation 8
The Beginning of A Significant Change The Beginning of A Significant Change to Chromatography <621>to Chromatography <621>� Because Waters is THE acknowledged leader in separation science, we were asked (by a customer) to help modernize LC column selection in General Chapter <621> Chromatography
� In the summer of 2009 Dr. Uwe Neue et. al. wrote a paper (article) that basically describes UPLC technology and method transfer– Dr. Neue’s calculations are also the basis of the ACQUITY UPLC
Columns Calculator
©2012 Waters Corporation 9
What Does This Stimuli Article What Does This Stimuli Article Propose?Propose?
� Stimuli Article PF 35(6) [Nov-Dec 2009]
©2012 Waters Corporation 10
What Does This Stimuli Article What Does This Stimuli Article Propose?Propose?
Variable Allowable ChangesCurrent Rgulation Stimuli Article Proposal
Particle Size -50% L/dp = Cst (±25%)Column Length ±70%Flow Rate ±50% Accordingly to L, dp and column IDColumn ID Any allowed Any allowed
Injection Volume Any reduction Any allowedColumn Temperature ±10% ±10%
Mobile Phase pH ±0.2 unit ±0.2 unit
If Stimuli Article PF 35(6) [Nov-Dec 2009] Officially Becomes Part of Chromatography <621>, Analysts Will Have More
Flexibility to Utilize Modern Chromatographic Techniques Such as UPLC Technology
If Stimuli Article PF 35(6) [Nov-Dec 2009] Officially Becomes Part of Chromatography <621>, Analysts Will Have More
Flexibility to Utilize Modern Chromatographic Techniques Such as UPLC Technology
Be aware that selectivity is not affected!This is only about geometrical transfer
©2012 Waters Corporation 11
USPUSP--NF Development ProcessNF Development Process
31st July, 2011Comment Period
EndedExpert Committee Discussions Began
Nov-Dec 2009Waters Stimuli SubmissionPF 35(6)
2nd May, 2011Amended
Chromatograhy<621> Posted in
PF 37(3)
*Deferrals are items that have been proposed in Pharmacopeial Forum (PF) but are not yet approved by an Expert Committee
1st March, 2012Revised Proposal
Posted inPF 38(2)
29th Dec, 2011Chromatography <621> System Suitability Deferred*
©2012 Waters Corporation 12
What Does This Revision Status What Does This Revision Status Change Mean?Change Mean?� How do you behave concerning revalidation?– Do you NEVER revalidate (never change methods) OR– Do you ALWAYS revalidate (even though changes were within USP allowable adjustments – audit fears) OR
– Do you SOMETIMES revalidate (when changes are outside of USP allowable adjustments)
� It means:– Sufficient changes to revised General Chapter <621> Chromatography were made that require public comment (again)
– Comment period will end 01-June-2012 according to PF 38(2)– Changes to <621> may occur late 2012 or 2013
� Depending of what is finally accepted, this will ease or remove the revalidation steps when moving to UPLC/UHPLC technologies– Assuming changes are allowed and system suitability requirements
are met
©2012 Waters Corporation 13
Lets Take a Concrete ExampleLets Take a Concrete Example
Geometrical Transfer Allowed if <621> will be
updatedL/dp = 29’412
Original HPLC MethodL/dp = 30’000
Geometrical Transfer Allowedwith Current Regulation
L/dp = 28’571
Geometrical Transfer Allowedwith Current Regulation
L/dp = 30’000
What are we doing until
the finalized USP Chapter
<621> revision?
©2012 Waters Corporation 14
Industry Trends:Industry Trends:The Market has ChangedThe Market has ChangedTransitioning from HPLC to UPLC Technology
� Increasing number of organizations have realized the business and scientific advantages of UPLC Technology
� Increased availability of UHPLC instruments and sub 3 µm chemistries provides vendor choice
� Technology shift has led companies to evaluate how to best utilize their existing HPLC instruments as they continue to invest in, and transition to, newer UPLC systems
During this transition, a number of challenges have arisen
that need to be addressed
©2012 Waters Corporation 15
Introducing The Introducing The Waters Waters 2.5 µm 2.5 µm eeXXtendedtended PPerformance erformance
Column FamilyColumn Family
©2012 Waters Corporation 16
� Packed in ultra-low dispersion hardware to minimize band spreading
� Designed to withstand high pressure– 4.6 mm ID capable of 9,000 PSI– 2.1 and 3.0 mm IDs compatible with UPLC pressures
� Flexibility in configurations– 2.1, 3.0 and 4.6 mm ID (2.1 and 3.0mm incorporating eCord™ technology)
– 30, 50, 75 and 100 mm lengths– 14 scalable stationary phases
� Packed with XBridge [BEH] and XSelect [CSH and HSS] 2.5 µm particles and chemistries– BEH C18, Shield RP18, C8, Phenyl, HILIC and Amide
– CSH C18, Phenyl-Hexyl and Fluoro-Phenyl– HSS C18, T3, C18 SB, Cyano and PFP
2.5 µm 2.5 µm eeXXtendedtended PPerformance Columns:erformance Columns:Product ScopeProduct Scope
What are the differences bewteenregular 2.5µm HPLC columns and 2.5µm XP columns ?
• 2.5µm XP can withstandhigher pressure (max pressure for 2.5µm is 6,000 PSI)• 2.5µm XP columns are fullyscalable from UPLC to prep• 2.5µm XP are packed in UPLC column harware• 2.1 and 3.0 mm ID 2.5µm XP columns have e-cord
©2012 Waters Corporation 17
Based Upon Two FullyBased Upon Two Fully--Scalable LC Scalable LC Column PlatformsColumn Platforms
Family designed and optimized for pH stability
Most MS-compatible HPLC columns on the market
Family designed and optimized for selectivity
Multiple particle substrates to solve multiple chromatographic
problems
1.7 [UPLC], 2.5, 3.5, 5 and 10 µm1.8 [UPLC], 2.5, 3.5 and 5 µm HSS1.7 [UPLC], 2.5, 3.5 and 5 µm CSH
New
2.5 µm XP eXtended
Performance Columns
©2012 Waters Corporation 18
Matching the Correct Column & Matching the Correct Column & SystemSystem� Two compendial (USP) method transfer examples
1. Isocratic Assay: Levonorgestrel and Ethinyl Estradiol– Compare original HPLC method with:
o XP column methods on Alliance HPLC systemo XP column method on ACQUITY UPLC H-Class systemo ACQUITY UPLC column method on ACQUITY UPLC H-Class system
2. Gradient Impurities: Abacavir– Transfer HPLC method to XP columns run on Alliance HPLC and ACQUITY UPLC H-Class systems
– Observe the effects of system band spread on separation and resolution
©2012 Waters Corporation 19
Positioning the Right Column with the Positioning the Right Column with the Right SystemRight System
System HPLC UPLCParticle Size 3.5 µm, 5 µm 2.5 µm XP 1.7/1.8 µm UPLC,
2.5 um XP, 3.5 µm, 5 µm1.7/1.8 µm UPLC,
2.5 um XP, 3.5 µm, 5 µmRoutine Pressure < 4000 psi < 4000 psi < 15000 psi (H-Class) < 18000 psi (I-Class)
Column ID 4.6 mm 4.6 mm 2.1 & 3.0 mm 1.0 & 2.1 mmColumn Length ≤ 250 mm ≤ 75 mm ≤ 150 mm ≤ 150 mm
©2012 Waters Corporation 20
Original Isocratic USP Assay: Original Isocratic USP Assay: 4.6 x 150 mm, 5 4.6 x 150 mm, 5 µµm (Alliance HPLC)m (Alliance HPLC)
AU
0.000
0.010
0.020
0.030
0.040
0.050
0.060
0.070
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
Flow Rate: 1.00 mL/minPressure: 1400 psiRun time: 8.00 minUSP Res: 6.9
levon
orge
strel
ethin
yles
tradio
l
System Suitability RequirementsUSP Resolution: NLT 2.5Peak Area Precision: NMT 2.0% RSD
L/dp = 30000N = 1XRs = 1X
Run time = 1XPressure = 1X
©2012 Waters Corporation 21
Compiled Status Compiled Status –– Isocratic MethodIsocratic MethodCriteria USP Res>2.5Criteria USP Res>2.5LC
ModeColumn
ID(mm)
ColumnLength(mm)
ParticleSize(µm)
RatioL/dp
FlowRate
(ml/min)Pressure(PSI)
RetentionTime(min)
USPRes.
Instrument Comments
HPLC 4.6 150 5 30’000 1 1400 8 6.9 Alliance OriginalHPLCMethod
HPLC 4.6 75 2.5 30’000 1 2600 4 6.5 Alliance USP Compliant w/ Current Guidelines
HPLC 4.6 50 2.5 20’000 2 3600 1.3 5 Alliance Not USP Compliantw/ Current Guidelines
HPLC 4.6 50 2.5 20’000 1.5 3050 1.6 5.2 Alliance USP Compliant w/ Current Guidelines
HPLC 2.1 75 2.5 30’000 0.42 4400 2 7.9 ACQUITYH-Class
Not USP Compliantw/ Current Guidelines
UPLC 2.1 50 1.7 29’400 0.61 7700 0.9 7 ACQUITYH-Class
Not USP Compliantw/ Current Guidelines
©2012 Waters Corporation 22
AU
0.000
0.010
0.020
0.030
0.040
0.050
0.060
Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40
AU
0.000
0.010
0.020
0.030
0.040
0.050
0.060
0.070
Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Isocratic Method Transferred to Isocratic Method Transferred to 4.6 mm XP Columns (Alliance HPLC)4.6 mm XP Columns (Alliance HPLC)
4.6 x 75 mm XP 2.5 µmFlow Rate: 1.00 mL/minPressure: 2600 psiRun time: 4.00 minUSP Res: 6.5
levon
orge
strel
ethin
yl es
tradio
l
Could not run at SCALED flow rate due to system pressure limitations
Results:Rs = 0.9X
Run time = 0.5XPressure = 2X
4.6 x 50 mm XP 2.5 µmFlow Rate: 2.00 mL/minPressure: 3600 psiRun time: 1.30 minUSP Res: 5.0
levon
orge
strel
ethin
yles
tradio
l LOWER L/dp column (20000) to reduce pressure:
Results:Rs = 0.7X
Run time = 0.16XPressure = 2.9X
> 50% flow rate change not <621>
compliant
©2012 Waters Corporation 23
Compiled Status Compiled Status –– Isocratic MethodIsocratic MethodCriteria USP Res>2.5Criteria USP Res>2.5LC
ModeColumn
ID(mm)
ColumnLength(mm)
ParticleSize(µm)
RatioL/dp
FlowRate
(ml/min)Pressure(PSI)
RetentionTime(min)
USPRes.
Instrument Comments
HPLC 4.6 150 5 30’000 1 1400 8 6.9 Alliance OriginalHPLCMethod
HPLC 4.6 75 2.5 30’000 1 2600 4 6.5 Alliance USP Compliant w/ Current Guidelines
HPLC 4.6 50 2.5 20’000 2 3600 1.3 5 Alliance Not USP Compliantw/ Current Guidelines
HPLC 4.6 50 2.5 20’000 1.5 3050 1.6 5.2 Alliance USP Compliant w/ Current Guidelines
HPLC 2.1 75 2.5 30’000 0.42 4400 2 7.9 ACQUITYH-Class
Not USP Compliantw/ Current Guidelines
UPLC 2.1 50 1.7 29’400 0.61 7700 0.9 7 ACQUITYH-Class
Not USP Compliantw/ Current Guidelines
©2012 Waters Corporation 24
AU
-0.010
0.000
0.010
0.020
0.030
0.040
0.050
0.060
Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00
AU
0.000
0.010
0.020
0.030
0.040
0.050
0.060
0.070
Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Isocratic Method Transferred to Isocratic Method Transferred to 4.6 mm XP Columns (Alliance HPLC)4.6 mm XP Columns (Alliance HPLC)
4.6 x 75 mm XP 2.5 µmFlow Rate: 1.00 mL/minPressure: 2600 psiRun time: 4.00 minUSP Res: 6.5
levon
orge
strel
ethin
yl es
tradio
l
Could not run at SCALED flow rate due to system pressure limitations
Results:Rs = 0.9X
Run time = 0.5XPressure = 2X
4.6 x 50 mm XP 2.5 µmFlow Rate: 1.50 mL/minPressure: 3050 psiRun time: 1.60 minUSP Res: 5.2
levon
orge
strel
ethin
yles
tradio
lLOWER L/dp column (20000)
to reduce pressure:
Results:Rs = 0.8X
Run time = 0.2XPressure = 2.2X
Both flow rates are <621> compliant
©2012 Waters Corporation 25
Compiled Status Compiled Status –– Isocratic MethodIsocratic MethodCriteria USP Res>2.5Criteria USP Res>2.5LC
ModeColumn
ID(mm)
ColumnLength(mm)
ParticleSize(µm)
RatioL/dp
FlowRate
(ml/min)Pressure(PSI)
RetentionTime(min)
USPRes.
Instrument Comments
HPLC 4.6 150 5 30’000 1 1400 8 6.9 Alliance OriginalHPLCMethod
HPLC 4.6 75 2.5 30’000 1 2600 4 6.5 Alliance USP Compliant w/ Current Guidelines
HPLC 4.6 50 2.5 20’000 2 3600 1.3 5 Alliance Not USP Compliantw/ Current Guidelines
HPLC 4.6 50 2.5 20’000 1.5 3050 1.6 5.2 Alliance USP Compliant w/ Current Guidelines
HPLC 2.1 75 2.5 30’000 0.42 4400 2 7.9 ACQUITYH-Class
Not USP Compliantw/ Current Guidelines
UPLC 2.1 50 1.7 29’400 0.61 7700 0.9 7 ACQUITYH-Class
Not USP Compliantw/ Current Guidelines
©2012 Waters Corporation 26
LC Systems and Column Positioning:LC Systems and Column Positioning:Transfer to ACQUITY UPLC HTransfer to ACQUITY UPLC H--Class SystemClass System
System HPLC UPLCParticle Size 3.5 µm, 5 µm 2.5 µm XP 1.7/1.8 µm UPLC,
2.5 um XP, 3.5 µm, 5 µm1.7/1.8 µm UPLC,
2.5 um XP, 3.5 µm, 5 µmRoutine Pressure < 4000 psi < 4000 psi < 15000 psi (H-Class) < 18000 psi (I-Class)
Column ID 4.6 mm 4.6 mm 2.1 & 3.0 mm 1.0 & 2.1 mmColumn Length ≤ 250 mm ≤ 75 mm ≤ 150 mm ≤ 150 mm
©2012 Waters Corporation 27
AU
0.00
0.02
0.04
0.06
0.08
Minutes0.00 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 3.20 3.40 3.60
Isocratic Method Transferred to Isocratic Method Transferred to ACQUITY UPLC HACQUITY UPLC H--Class SystemClass System
2.1 x 75 mm XP, 2.5 µmFlow Rate: 0.5 mL/minPressure: 5200 psiRun Time: 1.80 minUSP Res: 7.9
levon
orge
strel
ethin
yles
tradio
l
AU
0.00
0.02
0.04
0.06
Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60
2.1 x 50 mm UPLC, 1.7 µm Flow Rate: 0.61 mL/minPressure: 7700 psiRun Time: 0.90 min.USP Res: 7.0
levon
orge
strel
ethin
yles
tradio
l
Results:Rs = 1X
Run time = 0.1XPressure = 5.5X
Results:Rs = 1.1X
Run time = 0.25XPressure = 3.1X
Maximum benefits obtained on
ACQUITY UPLC System
©2012 Waters Corporation 28
Compiled Status Compiled Status –– Isocratic MethodIsocratic MethodCriteria USP Res>2.5Criteria USP Res>2.5LC
ModeColumn
ID(mm)
ColumnLength(mm)
ParticleSize(µm)
RatioL/dp
FlowRate
(ml/min)Pressure(PSI)
RetentionTime(min)
USPRes.
Instrument Comments
HPLC 4.6 150 5 30’000 1 1400 8 6.9 Alliance OriginalHPLCMethod
HPLC 4.6 75 2.5 30’000 1 2600 4 6.5 Alliance USP Compliant w/ Current Guidelines
HPLC 4.6 50 2.5 20’000 2 3600 1.3 5 Alliance Not USP Compliantw/ Current Guidelines
HPLC 4.6 50 2.5 20’000 1.5 3050 1.6 5.2 Alliance USP Compliant w/ Current Guidelines
HPLC 2.1 75 2.5 30’000 0.5 5200 1.8 7.9 ACQUITYH-Class
USP Compliant w/ Current Guidelines
UPLC 2.1 50 1.7 29’400 0.61 7700 0.9 7 ACQUITYH-Class
Not USP Compliantw/ Current Guidelines
©2012 Waters Corporation 29
Matching the Correct Column & Matching the Correct Column & SystemSystem� Two compendial (USP) method transfers
1. Isocratic Assay: Levonorgestrel and Ethinyl Estradiol– Compare original HPLC method with:
o XP column methods on Alliance HPLC systemo XP column method on ACQUITY UPLC H-Class systemo ACQUITY UPLC column method on ACQUITY UPLC H-Class system
2. Gradient Impurities: Abacavir– Transfer HPLC method to XP columns run on Alliance HPLC and ACQUITY UPLC H-Class systems
– Observe the effects of system band spread on separation and resolution
©2012 Waters Corporation 30
Gradient USP Impurities Assay: Gradient USP Impurities Assay: AbacavirAbacavir Method TransferMethod Transfer
System HPLC UPLCParticle Size 3.5 µm, 5 µm 2.5 µm XP 1.7/1.8 µm UPLC,
2.5 um XP, 3.5 µm, 5 µm1.7/1.8 µm UPLC,
2.5 um XP, 3.5 µm, 5 µmRoutine Pressure < 4000 psi < 4000 psi < 15000 psi (H-Class) < 18000 psi (I-Class)
Column ID 4.6 mm 4.6 mm 2.1 & 3.0 mm 1.0 & 2.1 mmColumn Length ≤ 250 mm ≤ 75 mm ≤ 150 mm ≤ 150 mm
©2012 Waters Corporation 31
Original Gradient USP Impurities: Original Gradient USP Impurities: 4.6 x 150 mm 5 4.6 x 150 mm 5 µµm (Alliance HPLC)m (Alliance HPLC)
AU
-0.002
0.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
0.018
0.020
Minutes
12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00
abac
avir
desc
yclop
ropy
l aba
cavir
o-py
rimidi
neab
acav
ir
1R, 4
R tra
ns ab
acav
ir
System Suitability Requirements
USP Res: NLT 1.5Flow Rate: 1.00 mL/minMax Pressure: 2800 psiGradient time: 35 minUSP Res: 3.1
©2012 Waters Corporation 32
Compiled Status Compiled Status –– Gradient MethodGradient MethodCriteria USP Res>1.5Criteria USP Res>1.5LC
ModeColumn
ID(mm)
ColumnLength(mm)
ParticleSize(µm)
RatioL/dp
FlowRate
(ml/min)Pressure(PSI)
RetentionTime(min)
USPRes.
Instrument Comments
HPLC 4.6 150 5 30’000 1 2800 35 3.1 Alliance OriginalHPLCMethod
HPLC 2.1 75 2.5 30’000 0.42 7250 8.8 2.9 ACQUITYUPLCH-Class
Not USP Compliantw/ Current Guidelines
HPLC 2.1 75 2.5 30’000 0.21 3600 17 3.3 ACQUITYUPLCH-Class
Not USP Compliantw/ Current Guidelines
HPLC 2.1 75 2.5 30’000 0.21 3500 17 NA Alliance NA
©2012 Waters Corporation 33
AU
-0.002
0.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
0.018
0.020
Minutes3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20
abac
avir
desc
yclop
ropy
laba
cavir
o-py
rimidi
neab
acav
ir
1R, 4
R tra
ns ab
acav
ir
Gradient USP Impurities Method Transferred to Gradient USP Impurities Method Transferred to 2.1 mm ID Columns (UPLC H2.1 mm ID Columns (UPLC H--Class)Class)
2.1 x 75 mm XP, 2.5 µmFlow Rate: 0.5 mL/minMax Pressure: 8630 psiGradient time: 7.30 min
USP Res: 2.9 (0.94X)
Scaled (same) linear velocity as original HPLC separation
What if I ran this 2.1 mm ID XPcolumn on an Alliance HPLC
system?
©2012 Waters Corporation 34
Compiled Status Compiled Status –– Gradient MethodGradient MethodCriteria USP Res>1.5Criteria USP Res>1.5LC
ModeColumn
ID(mm)
ColumnLength(mm)
ParticleSize(µm)
RatioL/dp
FlowRate
(ml/min)Pressure(PSI)
RetentionTime(min)
USPRes.
Instrument Comments
HPLC 4.6 150 5 30’000 1 2800 35 3.1 Alliance OriginalHPLCMethod
HPLC 2.1 75 2.5 30’000 0.5 8630 7.3 2.9 ACQUITYUPLCH-Class
USP Compliant w/ Current Guidelines
HPLC 2.1 75 2.5 30’000 0.21 3600 17 3.3 ACQUITYUPLCH-Class
Not USP Compliantw/ Current Guidelines
HPLC 2.1 75 2.5 30’000 0.21 3500 17 NA Alliance NA
©2012 Waters Corporation 35
Running a 2.1 x 75 mm XP Column:Running a 2.1 x 75 mm XP Column:Alliance vs. ACQUITY UPLC HAlliance vs. ACQUITY UPLC H--ClassClass
AU
0.000
0.005
0.010
0.015
0.020
Minutes6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00
ACQUITY UPLC H-ClassFlow Rate: 0.21 mL/minMax Pressure: 3600 psiGradient Time: 17.0 minUSP Res: 3.3 (1.1X)
o-py
rimidi
neab
acav
ir
1R, 4
R tra
ns ab
acav
ir
desc
yclop
ropy
laba
cavir
abac
avir
Why the HUGE difference in performance?
AU
-0.005
0.000
0.005
0.010
0.015
0.020
Minutes
10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00
Alliance HPLCFlow Rate: 0.21 mL/minMax Pressure: 3500 psiGradient Time: 17.0 minUSP Res: ?
abac
avir
desc
yclop
ropy
laba
cavir
o-py
rimidi
neab
acav
ir
1R, 4
R tra
ns ab
acav
ir
Lower flow rate based upon HPLC system pressure limit
©2012 Waters Corporation 36
– Wide chromatographic bando Increased peak widtho Dilution effect decreases peak height and sensitivity
– Narrow chromatographic bando Decreased peak widtho High efficiency, sensitivity and resolution
What is Band Spreading?What is Band Spreading?� Any component of the chromatographic system [instrument
and column] that contributes to the distortion and broadening of a chromatographic band
Minimize band spreading to improve chromatographic performance
©2012 Waters Corporation 37
Waters Disruptive Technology is the Providing a Waters Disruptive Technology is the Providing a Complete (Holistic) Solutions for Meaningful Impact of Complete (Holistic) Solutions for Meaningful Impact of Achieving Theoretical Promise of SeparationAchieving Theoretical Promise of Separation
� All developments are focused on the separation integrity through system – Expanding and optimizing particle synthesis and packing of columns– Injector design, injector volume, fittings, flow path, sealing surfaces– Reduced tubing volumes– Optimized detectors to minimize dispersion and increasing signal while minimizing noise
22det
2,det
2,
2,
2,
2,
2, Fectorectorvpostcolumnvcolumnvprecolumnvinjectorvtotalv ⋅+++++= τσσσσσσ
Injectionvolume
+injectorband-
Spreading
Tubingbetweeninjectorand
column
Columnvolume
+frits
Tubingbetweencolumnand
detector
Band-spreadinginside thedetectorcell+
Tubing
Time-basedBand-
spreadingin the
Detector(SamplingRate; TimeConstant)
©2012 Waters Corporation 38
Summary: Positioning Column Summary: Positioning Column Configurations with LC SystemsConfigurations with LC Systems
Chromatographic System Band Spread* Recommended Column IDPrimary Secondary
Shimadzu Prominence UFLC 41 µL4.6 mm 3.0 mmAlliance 2695 HPLC 29 µL
Agilent 1260 UHPLC (600 bar) 28 µLThermo Accela UHPLC 21 µL 3.0 mm 2.1 mmAgilent 1290 UHPLC (1200 bar) 17 µLACQUITY UPLC 12 µL
2.1 mm 3.0 mmACQUITY UPLC H-Class w/Column Manager 12 µLACQUITY UPLC H-Class 9 µLACQUITY UPLC I-Class (FTN) 7.5 µL 2.1 mm 1.0 mmACQUITY UPLC I-Class (FL) 5.5 µL
(*) – measured by System Marketing Laboratory
©2012 Waters Corporation 39
Compiled Status Compiled Status –– Gradient MethodGradient MethodCriteria USP Res>1.5Criteria USP Res>1.5LC
ModeColumn
ID(mm)
ColumnLength(mm)
ParticleSize(µm)
RatioL/dp
FlowRate
(ml/min)Pressure(PSI)
RetentionTime(min)
USPRes.
Instrument Comments
HPLC 4.6 150 5 30’000 1 2800 35 3.1 Alliance OriginalHPLCMethod
HPLC 2.1 75 2.5 30’000 0.5 8630 7.3 2.9 ACQUITYUPLCH-Class
USP Compliant w/ Current Guidelines
HPLC 2.1 75 2.5 30’000 0.21 3600 17 3.3 ACQUITYUPLCH-Class
Not USP Compliantw/ Current Guidelines
HPLC 2.1 75 2.5 30’000 0.21 3500 17 NA Alliance NA
©2012 Waters Corporation 40
Method TransferMethod TransferSummarySummary� Methods can be transferred to 4.6 mm ID XP columns on HPLC systems– May need to reduce flow rate when using >50mm length columns– If column length and particle size are not scaled, resolution values
will be different than original
� Improved performance for 2.1 mm ID XP and UPLC columns obtained on lower dispersion, higher-pressure-tolerant ACQUITY UPLC systems– Properly scaled column lengths and flow rates can be utilized
� Maximum performance benefits with complete ACQUITY UPLC system solution
©2012 Waters Corporation 41
SummarySummary� Encouraging sign from regulatory agencies to facilitate adoption of new technology
� Waters will continue to work with USP and other regulatory bodies worldwide– We are committed to advance and modernize separation science
� USP Method Transfer Project gives us a keen understanding of challenges associated with running drug final formulations
� 2.5 µm XP particles is a ready solution while USP Chapter is being updated– XP columns bridge the gap between HPLC and UPLC– Positioned with our UPLC system, productivity could be significantly improved
today– Methods can be modernized NOW within existing compendial guidelines– It offers scalability to real UPLC– H-Class is definitively the best LC for today and tomorrow since new R&D project
are being developed on 1,7 µm particles
©2012 Waters Corporation 42
AU
0.00
0.10
0.20
0.30
Minutes0.00 4.00 8.00 12.00 16.00 20.00 24.00 28.00
XSelect CSH C184.6 x 100 mm, 3.5 µmHPLC System
1ml/min3.5 µmTo
lmetin
Naprox
en
Feno
profen
Indom
etha
cin Diclo
fenac
AU
0.00
0.10
0.20
0.30
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00
XSelect CSH C18 XP4.6 x 75 mm, 2.5 µm
HPLC SystemHPLC Conditions due to H/W
pressure limitation1,4ml/min
2X Faster than 3.5 µm HPLC
Concrete Summary ExampleConcrete Summary Example
ACQUITY UPLC CSH C182.1 x 50 mm, 1.7 µmUPLC System
0.8ml/min
9X Faster than 3.5 µm HPLC
AU
0.00
0.10
0.20
0.30
0.00 0.50 1.00 1.50 2.50 3.00 3.50Minutes 2.10
1.7 µm
5X Faster than 2.5 µm HPLC
XSelect CSH C18 XP2.1 x 75 mm, 2.5 µm
UPLC SystemWith UPLC Conditions
Opt. Flow Rate: 0.54mL/min
AU
0.00
0.05
0.10
0.15
0.20
0.25
0.30
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.004.50
Tolm
etin
Naprox
en
Feno
profen
Indom
etha
cin
Diclo
fenac 2.5 µm XP
+20%
+20%
2.5 µm XP
©2012 Waters Corporation 43
Questions?Questions?
For how many years do you envisage the system that you are purchasing remaining “fit for
purpose”
©2012 Waters Corporation 44
State of System State of System LifeCycleLifeCycle
Year 2012Run existing legacy HPLC
methods
Year 2016Technology in hand
fully optimized
Year 2020State of the art technology fully
deployed
©2012 Waters Corporation 45
ACQUITY UPLC I-Class•New chemical entities, research, impurity analysis•1.7/1.8 µm UPLC columns
Investment Protection Approach…Investment Protection Approach…Pe
rfor
man
ce L
evel
Future-Proofing [Long-Term Vision/Strategy]
ACQUITY UPLC H-Class•Method development, method transfer, QA/QC•Changing priorities from small – to – large molecules•1.7/1.8 µm UPLC columns as well as 2.5 µm XP, 3.5 and 5 µm HPLC columns
Alliance 2695•Faster HPLC assays•2.5 µm XP columns
•Traditional HPLC assays•3.5 / 5 µm HPLC columns
©2012 Waters Corporation 47
What Happen at the European LevelWhat Happen at the European Level� Will EurP follow USP?
– USP and EurP are harmonizing both chapter <621> and 2.2.46 to the extent possible
� What about the EurP Chapter 2.2.46?– Draft 2.2.46 chapter has already been written
� When will this be released?– EurP expect to release the new 2.2.46 chapter at the same time as
the USP <621> or with a short delay