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©2007 Waters Corporation

Pharmaceutical Solutions Pharmaceutical Solutions With UltraPerformance LCWith UltraPerformance LC®®

©2007 Waters Corporation

Evaluation Form

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©2007 Waters Corporation 3

Requirements for High Requirements for High Resolution SeparationsResolution Separations

Liquid Chromatography and LC/MS are key in the pharmaceutical business developmentLC and LC/MS is used in the areas of high throughput screening, impurity profiling, metabolite ID, bioanalysis, quality control and product releaseThese activities are critical to the development of new drug productsIn these analysis there is a need for rapid, high resolution separations to ensure:-—The detection of all of the analytes in the sample—High throughput for productivity—Maximization of sensitivity


Analytical ChallengesAnalytical Challenges

Simple rapid development of analysis

High resolution separations to ensure the full characterization of samples

Easy simple transfer from HPLC to UPLC®

Fast sensitive detectors

Robust analysis


Improving ChromatographyImproving ChromatographyBenefits ofBenefits of UPLCUPLC®®

Analyte detection in LC and LC/MS depends upon a high quality LC separation

OptionsOptions— LC resolution can be improved by increasing column length

or reducing particle size

— Column efficient increases with the square root of the column length, but is inversely proportional to particle size

— Small particles give increased performance with reduced analysis time

— Increased column length results in longer analysis times

The best choice is to use smaller particles and The best choice is to use smaller particles and shorter columns. With the exception of chiral.shorter columns. With the exception of chiral.


ACQUITY Ultra Performance LCACQUITY Ultra Performance LC®®

UPLCUPLC®®

UPLC (Ultra Performance LC) leverages the theories and principles of HPLC with sub 2μm particles

Generates ultra high resolution chromatograms

Improved Chromatographic efficiency results in more sensitive analysis

Requires a LC system with very low system volumes

Give rise to fast analysis for increased throughput, sensitivity and peak capacity


Smaller ParticlesSmaller Particlesthe enabler of productivitythe enabler of productivity

6

Optimum combination of

speed, resolution and sensitivity

2

4

68

10

12

14

16

18

20

22

24

26

28

30

0 0.5 1 1.5 2 2.5 3 3.5 4

Linear Velocity u (mm/sec)

H (µ

m)

5 µm Particle

HETP

Existing HPLCTechnology

5

Rs = N4 ( α -1

α ) kk+1( )

RetentivitySelectivitySystemEfficiency

Rs = N4N4 ( α -1

α ) kk+1( )

RetentivitySelectivitySystemEfficiency

Efficiency is the key to easier method development faster analysis and more sensitive detection


Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00

UPLCUPLC®® Method OptionsMethod Optionsincreased resolution or same resolution fasterincreased resolution or same resolution faster

2.1 x 150 mm, 5 µmRs (2,3) = 4.29

12 3

HPLC

20.00

0.26

Abs

orba

nce

at 2

70 n

m

0.00

Minutes0.40 0.80 1.20 1.60 2.00 2.50

2.1 x 50 mm, 1.7 µmRs (2,3) = 4.281

23

8X Speed3.4X SensitivitySame Resolution

0.26

Abs

orba

nce

at 2

70 n

m

0.00

UPLC

Faster, More Sensitive Methods

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

3

2.1 x 100 mm, 1.7 µmRs (2,3) = 6.38

1

2

4.5X Speed2X Sensitivity1.5X Resolution

4.50

0.26

Abs

orba

nce

at 2

70 n

m

0.00

UPLC

Faster, More Sensitive, Higher Resolution Methods


Transfer of a LC MethodTransfer of a LC MethodHPLC to UPLCHPLC to UPLC®®

Transferring of a method from HPLC to UPLC®

involves a few simple steps:-—Scaling of the column geometry

—Scaling of the flow rate to match the new particle size

—Selection of method required, same efficiency, maximum efficiency, fastest analysis

This can be done manually or with the ACQUITY UPLC® Calculator

Giving simple easy method transfer


HPLC Separation of SimvastatinHPLC Separation of Simvastatin

O

OO

OOH

CH3 CH3 CH3

CH3

CH3H

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

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

Efficiency = 12112


Transfer of Method to UPLCTransfer of Method to UPLC®®


ACQIUTY UPLCACQIUTY UPLC®®

rapid simple method transferrapid simple method transfer

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

Efficiency = 17685

Maximum EfficiencyMaximum Efficiency Equal EfficiencyEqual Efficiency Fastest AnalysisFastest Analysis

Efficiency = 12874 Efficiency = 977


Impurity ProfilingImpurity ProfilingRanitidine Ranitidine

Ranitidine HCl, Zantac®

Ranitidine is in a class of drugs called histamine receptor antagonists. Ranitidine works by decreasing the amount of acid the stomach produces

Aim of this study was to develop a MS compatible UPLC method for the analysis of the impurities in ranitidine

Compare performance to that of traditional HPLC


CH3NH

O

ONH

N SCH3

CH3

N+O-

O

S - oxide [M+H] = 331.1440 (C)

N+

O

O-

O

NH

NH

N

S

CH3 CH3

CH3

Ranitidine [M+H] = 315.1491 (active)

R - ethanamine [M+H] = 215.1218 (F)

O NH2

N

S

CH3 CH3

O

N+

O

O-

O

NH

N

S

CH3 CH3

R-nitroacetamide [M+H] = 301.1174 (E)

CH3

NH

ONH

N S

CH3 CH3

N+O-

O

O

N - oxide [M+H] = 331.1440

CH3

OH

N

NH

S

N

R - one oxime [M+H] = 160.0544 (A)

CH3

NH

NH

S

CH3N

CH3

O

N+

O-

O

CH3

NH

NH

S

CH3N

CH3

O

N+

O-

Oadduct [M+H] = 641.2903 (J)

N,N bis [M+H] = 498.2208

O

NH

N

S

CH3 CH3

N+ O

-

O

2

RanitidineRanitidineknown impuritiesknown impurities


HPLC ChromatogramHPLC ChromatogramRanitidine ImpuritiesRanitidine Impurities

AU

-0.010

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.080

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

A

B

C

DE

F

G - Active

H

I

J

*K

AU

-0.010

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.080

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

A

B

C

DE

F

G - Active

H

I

J

*K


Method DevelopmentMethod Developmentreversedreversed--phase column selectivity chartphase column selectivity chart

(ln [k] acenaphthene)

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

YMC-Pack™ ODS-A™

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–H80

YMC J'sphere™ ODS–M80

Inertsil® CN-3

Waters Spherisorb® S5CN

Nova-Pak® CN HP

SymmetryShield™ RP8

SymmetryShield™ RP18XTerra® RP8

XTerra® RP18

-0.6-0.3

0

0.3

0.60.9

1.2

1.51.8

2.12.42.7

33.3

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 C8Luna ®C18 (2)

ACQUITY UPLC™BEH C18

XTerra ®Phenyl Luna ™

Phenyl Hexyl

ChromolithTM RP-18 Atlantis® dC18

Zorbax® XDB C18ACT Ace® C18

Zorbax® SB C18

SunFire ™ C8

Luna®C8 (2)

ACQUITY UPLC™Shield RP18

ACQUITY UPLC™BEH C8

ACQUITY UPLC™

BEH Phenyl


Choosing The Best Column for SeparationChoosing The Best Column for SeparationACQUITY UPLCACQUITY UPLC®® ColumnsColumns

ACQUITY UPLC® BEH C18

ACQUITY UPLC ® BEH C8

ACQUITY UPLC ® BEH Shield RP18

ACQUITY UPLC™ BEH Phenyl

USP L11

USP L1

USP L7

USP L1

Revisions are currently under way to formally include 1.7μm particles in the USP listings


ACQUITY UPLCACQUITY UPLC®® Column ManagerColumn Manager

Thermostatted from 10 - 90°C

Automated switching among four columns and a bypass channel

eCord™ Information Management for each column

Accepts column sizes from 2.1x 30mm to 4.6 x 150mm

Passive pre-column solvent heating and post-column cooling


ACQUITY UPLCACQUITY UPLC®® System with column System with column manager and SQD,TUV and ELSDmanager and SQD,TUV and ELSD


ACQUITYACQUITY®® Column ManagerColumn ManagerFour 2.1 x 50mm columns installedFour 2.1 x 50mm columns installed


Column Manager Fluidic Column Manager Fluidic SchematicSchematic


Column Manager Column Manager –– instrument instrument methodmethod


AU

0.00

0.02

0.04

AU

0.00

0.02

0.04

AU

0.00

0.02

0.04

AU

0.00

0.02

0.04

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

BEH™ Phenyl

BEH™ C18

BEH™ Shield

BEH™ C8

A FE

CD

B

API

H I

J

C

C

CI

I, J

I

F, API

D

D

D

B

Column Chemistry ComparisonsColumn Chemistry ComparisonsUPLCUPLC®® 55--95% scouting gradients95% scouting gradients


AU

0.00

0.05

0.10

0.15

0.20

0.25

AU

-0.20

-0.10

0.00

0.10

0.20

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

pH 9 20mM ammonium bicarbonate

pH 520mM ammonium acetate

pH As a LC ParameterpH As a LC Parametercomparisonscomparisons


AU

0.000

0.010

0.020

0.030

0.040

0.050

0.060

AU

0.00

0.02

0.04

0.06

AU

0.00

0.02

0.04

0.06

Minutes

1.00 2.00 3.00 4.00 5.00 6.00 7.00

Temp = 400C

Temp = 300C

Temp = 500C

I + JBC

BC

BC

I J

I J

Effect of TemperatureEffect of TemperatureSelectivitySelectivity

Temperature Rs for (B + C) Rs for (I + J)

30 0C 1.80 0

40 0C 3.52 ~1

50 0C 5.33 1.54


Importance of Sampling RateImportance of Sampling Rate

Must ensure enough points are collected across a peak to adequately define the peak shape.

Peak detection algorithms require a minimum number of points across a peak to distinguish it from baseline noise and correctly determine peak lift off and touch down.

A peak which does not have enough data points will be difficult to integrate and therefore have irreproducible peak areas and heights.


AU

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

0.022

0.024

0.026

0.028

0.030

0.032

0.034

Minutes0.565 0.570 0.575 0.580 0.585 0.590 0.595 0.600 0.605 0.610 0.615 0.620 0.625 0.630

Effect of Sampling Rate on Peak Effect of Sampling Rate on Peak ShapeShape

1 pt/s2 pts/s5 pts/s10 pts/s20 pts/s40 pts/s


Optimizing Data CollectionRatesOptimizing Data CollectionRates

Data Rate

Points Across Peak

Peak Area %RSD

Peak Height %RSD

5 Hz 9 4.33 7.77

10 Hz 14 1.50 1.37

20 Hz 26 1.20 1.00

40 Hz 52 0.72 0.71

80 Hz 104 0.49 0.49

The data rate must be optimized to yields greater than 15 points across the peak to achieve reproducibility requirements while having the least impact on noise

5 Hz

40 Hz


Effect of Sampling Rate on Effect of Sampling Rate on ReproducibilityReproducibility

Sampling Sampling RateRate

Points Points Across Across PeakPeak

Peak Peak Area Area %RSD%RSD

Peak Peak Height Height %RSD%RSD

1 pt/s 2 2.436 15.515

2 pts/s 4 1.790 13.455

5 pts/s 7 0.971 3.962

10 pts/s 13 1.129 1.015

20 pts/s 25 0.603 1.156

40 pts/s 49 0.284 1.127


MS with UPLCMS with UPLC®®

The use of MS increases peak specificity, SIR, MRM

In the majority of cases MS can increase sensitivity

The spectral pattern can be used to confirm the identity of analytes

MS spectra can be used to help withpeak tracking in method development.

Like other detectors the MS must be able collect data at a fast enough toensure the peak shape is maintained


Time0.10 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 1.60 1.70 1.80 1.90 2.00

%

0

100acq11mix6 1: Scan ES+

BPI1.31e8

1.05

0.74

0.58

0.24

0.680.64

1.00

1.22

1.26

1.41

Peak at 1.41 min 1.5 sec at baseline10000da/sec 23 scans across peak

Terfe

nadi

ne

Oxy

buty

nin

Trim

ipra

min

e

Dip

henh

ydra

min

e

Pro

pran

olol

Lido

cain

eD

oxyl

amin

e

Caf

fein

e

Eph

edrin

e

ACQUITYACQUITY®® SQD Maintaining Peak FidelitySQD Maintaining Peak FidelityHigh Scan Rate MSHigh Scan Rate MS

1.400 22275731.401 26256891.403 32623071.404 47053201.405 66985011.406 84895641.407 94074641.408 105724101.410 127896701.411 150318611.412 165974851.413 169260881.414 161593141.415 129845721.417 101603621.418 86540481.419 85601261.420 69053511.421 55428281.422 45199571.424 41617671.425 32353271.426 2770925

150-650 50ms scan time


Pos/Neg SwitchingPos/Neg Switching5,000 da/sec Vs.10,000 da/sec5,000 da/sec Vs.10,000 da/sec

10000am u 20isd cont

Tim e0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20

%

0

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20

%

0

100analgesics23 1: Scan ES+

BPI5.40e7

1.36

1.20

1.050.84

0.050.21 0.68

0.94

1.751.601.78

analgesics26 1: Scan ES+ BPI

3.47e71.36

1.20

1.060.95

0.85

0.790.04 0.08 0.401.981.731.541.47 1.89

2.02 2.132.19 2.33

100-1100 dapos neg sw itching 5000da/sec 100m s ISD5 poin ts across peak at 1.06 m in

100-1100 da pos neg sw itching 10000da/sec 20m s ISD13 ponts across peak at 1.06 m in

1.038 22049281.048 58626561.059 146688001.069 83345921.079 3900160

1.032 13208961.036 11488641.040 14314881.045 78520321.049 183490561.053 200591361.057 181145601.061 192512001.065 153640961.069 102236161.073 101939201.077 59299841.081 3297280


Inte

nsity

0

1x107

2x107

3x107

4x107

5x107

AU

0.00

0.05

0.10

0.15

0.20

0.25

Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00

ES+ MS TIC

PDA-UV (230nm)

Increase Sensitivity and Peak TrackingIncrease Sensitivity and Peak Trackingsingle quad MSsingle quad MS


Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00

Increase Sensitivity and Peak TrackingIncrease Sensitivity and Peak Trackingsingle quad MSsingle quad MS


Spectrum IndexSpectrum IndexSpectrum Index Plot

SampleName BEH C18 pH9 20mM C18drylab; Vial 1:F,1; Injection 1; Date Acquired 5/11/2005 4:28:37 PM

m/z200.00

600.00

159.94 0.74

0.739m/z

200.00

600.00

290.02 1.10

1.105m/z

200.00

600.00

329.19 1.65

1.651m/z

200.00

600.00

387.072.65

2.645m/z

200.00

600.00

331.11 2.80

2.796m/z

200.00

600.00

155.96 3.09

3.091m/z

200.00

600.00

256.08

300.05 3.60

3.599m/z

200.00

600.00

215.14 4.15

4.144m/z

200.00

600.00

315.17 4.41

4.409m/z

200.00

600.00

170.93

215.97

241.24

272.15286.17

4.79

4.788m/z

200.00

600.00

329.19 5.22

5.223m/z

200.00

600.00

314.76 5.56

5.564m/z

200.00

600.00

297.16 5.68

5.684

Inte

nsity

0

1x107

2x107

3x107

4x107

5x107

6x107

Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00

0.73

9

1.10

5

1.65

1 2.64

52.

796

3.09

1

3.59

9

4.14

4

4.40

9

4.78

8

5.22

3

5.56

45.

684

5.76

9

6.58

1

Inte

nsity

0

1x107

2x107

3x107

4x107

5x107

6x107

Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00

Known Impurities that could be verifieda. 5,6-dihydro-3-methylamino-2H-1,4-thiazin-

2-one oxime (S,D) [M+H] = 160.0544

b. N-[2-[[[5-[(dimethylamino)methyl]-2-furanyl]thio]ethyl]-N’-methyl -2-nitro-2,2-ethenediamine-N-oxide (D) [M+H] = 331.1440; 315.1491

c. N-[2-[[[5-[(dimethylamino)methyl]-2-furanyl]sulphinyl]ethyl]-N’-methyl -2-nitro-1,1-ethenediamine (D) [M+H] = 331.1440

d. N-[2-[[[5-[(dimethylamino)methyl]-2-furanyl]methyl]thio]ethyl]-2-nitroacetamide (D) [M+H] = 302.1174

e. 5-[[(2-aminoethyl)thio]methyl]-N,N-dimethyl-2-furanmethanamine (S,D) [M+H] = 215.1218

f. Ranitidine formaldehyde adduct (C27H44N8O6S2) (D) [M+H] = 641.2903

g. N,N’bis[2-[[[5-[(dimethylamino)methyl]-2-furanyl]methyl]thio]ethyl]-2-nitro-1,1-ethenediamine (S) [M+H] = 498.2208

As it can be seen, there are also many un-identified peaks that need further characterization using either exact mass Tof data, MS/MS data, and/or NMR data


Final MethodFinal MethodRanitidine Impurity ProfileRanitidine Impurity Profile

AU

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00

d

a

f

e

gb c

m

j k lh

n

i

o

RanitidineConditionsColumn: ACQUITY UPLC BEH C18Dimensions: 50 x 2.1, 1.7um Mobile Phase A: 20mM Ammonium BicarbonateMobile Phase B: MethanolWeak Wash : 95:5 Water: MeOH 1200uLStrong Wash : 50:50 Water: MeOH 300uLFlow Rate: 0.7 mL/minGradient: Time Profile

(min) %A %B 0.0 99.0 1.01.0 99.0 1.02.0 85.0 15.06.0 30.0 70.0

Injection Volume: 1.0 µLTemperature: 30oCDetection: UV @ 245 nm


Nominal MassHPLC

ResolutionUPLC

Resolution Fold Difference

160

176 1.90 3.14 1.7

231 0.83 1.16 1.4

290 0.62 1.16 1.9

300 1.50 1.05 0.7

329 2.72 5.96 2.2

272 0.84 13.03 15.5

231 0.83 1.56 1.9

387 0.50 2.90 5.8

156 2.22 7.72 3.5

331 3.15 4.16 1.3

287 0.50 4.40 8.8

256,300 2.09 3.17 1.5

215 2.27 11.51 5.1

(active) 315 3.05 5.50 1.8

286 0.24 7.87 32.8

284 1.39 3.34 2.4

329 3.49 8.77 2.5

314,628 1.04 5.68 5.5

452 0.30 3.50 11.7

297 2.72 1.13 0.4

*321, 641 2.55 2.88 1.1

Average 5.2

Resolution BenefitsResolution BenefitsHPLC vs. UPLCHPLC vs. UPLC®®


Nominal Mass HPLC - UV S/N UPLC -UV S/N Fold Difference

160 48.0 511.9 10.7

176 6.9 27.1 3.9

231 2.5 42.3 16.9

290 13.1 110.4 8.4

300 1.6 29.4 18.2

329 19.6 59.6 3.0

272 19.6 40.6 2.1

231 12.0 23.5 1.9

387 62.1 185.6 3.0

156 74.2 209.0 2.8

331 172.3 600.6 3.5

287 4.5 18.3 4.1

256,300 85.7 157.8 1.8

215 165.6 221.8 1.3

(active) 315 13081.3 20278.9 1.6

286 13.6 30.1 2.2

284 17.7 23.8 1.3

329 82.6 154.9 1.9

314,628 8.3 39.3 4.8

452 5.4 26.6 4.9

297 110.9 185.2 1.7

*321, 641 1127.8 2451.2 2.2

Average 4.6

Sensitivity BenefitsSensitivity BenefitsHPLC vs. UPLCHPLC vs. UPLC®®


Drug Substance AnalysisDrug Substance AnalysisBudesonide Impurity ProfileBudesonide Impurity Profile

Budesonide , Pulmicort

Budesonide is in a class of drugs called corticosteroids. Budesonide works in the intestines to reduce inflammation, tissue damage, and diarrhoea.

Initial HPLC methodology generated required a 30 minute analysis time

Aim of this study was to develop a UPLC®(MS) method for the analysis of the impurities in budesonide

Compare performance to that of traditional HPLC


Structure of Budesonide and Structure of Budesonide and known related substancesknown related substances

(*) Denotes epimeric form

OHCH3

OH

O

OH

OH

CH3

O

H H

HImpurity A[M+H] = 377.1964

OCH3

O

O

O

OH

CH3

CH3

O

H H

H

H

Impurity D[M+H] = 429.2277

OCH3

O

O

OH

OH

CH3

CH3

O

H H

H

H

Budesonide[M+H] = 431.2434

* CH3

HO

CH3

O

O

OH

OH

CH3

O

H H

H

Impurity F[M+H] = 403.2120

*

OCH3

O

O

OH

OH

CH3

CH3

O

H

H

H

Impurity E[M+H] = 429.2277

*

H

H

O OHCH3

O

O

OHCH3

CH3

O

H

H

Impurity C[M+H] = 431.2433

*

OCH3

O

O CH3

OH

OH

CH3

O

H H

H

H

Impurity B[M+H] = 403.2120

*

OCH3

O

O

OH

OH

CH3

CH3

O

H H

H

H

Impurity G[M+H] = 433.2512

*


Column ScoutColumn ScoutBudesonideBudesonide

ACQUITY TUV ChA - ACQUITY TUV ChA 240nm

AU

0.00

0.20

0.40

0.60


AU

0.00

0.20

0.40


AU

0.00

0.20

0.40

0.60

0.80


AU

0.00

0.20

0.40

0.60

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

BEH Phenyl

BEH C18

BEH Shield

HSS C18



AU

0.000

0.005

0.010

0.015


AU

0.000

0.005

0.010

0.015


AU

0.000

0.005

0.010

0.015


AU

0.000

0.005

0.010

0.015

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

BEH Phenyl

BEH C18

BEH Shield

HSS C18

Column Scout Column Scout Budesonide(zoomed view)Budesonide(zoomed view)


Temperature ScoutTemperature Scoutreduced analysis timereduced analysis time

240.0nm - PDA Spectrum - PDA Spectrum (230-350)nmAU

0.000

0.005

0.010

0.015

0.020

240.0nm - PDA Spectrum - PDA Spectrum (230-350)nm

AU

0.000

0.005

0.010

0.015

0.020

240.0nm - PDA Spectrum - PDA Spectrum (230-350)nm

AU

0.000

0.005

0.010

0.015

0.020

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

300C

400C

600C

Resolution between epimers lostResolution between epimers lost


Budesonide Final MethodBudesonide Final MethodAU

0.000

0.007

0.014

0.021

0.028

Minutes0.00 1.10 2.20 3.30 4.40 5.50 6.60 7.70 8.80 9.90

0.00

0.70

1.40

0.00 3.00 6.00

R-epimer

S-epimer

6

7

4

3

2

1

5

Name Retention Time

Resolution SymmetryFactor

Signal/Noise EPPlates

R— epimer 5.073 N/A 1.05 10262 17011

S— epimer 5.476 2.46 1.02 6646 17390

EP HPLC method requirements met.


LC/MS Trace ComparisonsLC/MS Trace Comparisons4 lots of budesonide4 lots of budesonide

spectrum

Time0.50 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 7.50 8.00 8.50 9.00

%

0

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 7.50 8.00 8.50 9.00

%

0

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 7.50 8.00 8.50 9.00

%

0

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 7.50 8.00 8.50 9.00

%

1

Budesonide Impurity 3 Sm (SG, 10x1) 1: Scan ES+ BPI

1.29e7x10

5.11

4.923.892.37

0.41 2.170.72 1.861.443.103.012.72 4.794.31 8.307.73 8.58 8.90


4.16e7x10

5.12

4.90 7.19 7.75


4.32e7x10 5.12

4.913.882.760.50 2.93


4.61e7x10

5.12

3.080.72 7.21

6.887.77

Supplier B

Supplier A

Supplier C

Supplier D


EP Related Substances TestEP Related Substances Test

European Pharmacopoeia Related Substances Test

Specification

Supplier A Supplier B Supplier C Supplier C

Individual Impurities(x < 2.5µg/mL ∑ of epimers areas)

Fail Fail Pass Fail

Total Impurities(x < 7.5µg/mL ∑ of epimers areas)

Fail Fail Pass Fail

R—epimer/S—epimer Ratio(S—epimer is 40.0% to 51%

∑ of epimers areas)

59.24%/40.76%

50.49%/49.51

51.38%/48.62

58.66%/41.34

Purity 98.24% 97.99% 99.52% 98.07%


Individual Impurities Individual Impurities Specified Impurity Spectra of 429Specified Impurity Spectra of 429

OCH3

O

O

O

OH

CH3

CH3

O

H H

H

H

Impurity D[M+H] = 429.2277

OCH3

O

O

OH

OH

CH3

CH3

O

H

H

H

Impurity E[M+H] = 429.2277

*

OR

Could be..


Individual Impurities that failed for Spectrum EPIndividual Impurities that failed for Spectrum EPSpectra of Unknown/ Unspecified [M+H] = 435Spectra of Unknown/ Unspecified [M+H] = 435


Unknown mass 435.2184Unknown mass 435.2184

m/z100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750 775 800

%

0

100030806_PR_ESIPOS_015 248 (2.216) Cm (242:252-(261:275+183:209)) 1: TOF MS ES+

7.01e3435.2184

415.2125

399.2185357.1726

323.1668152.7430 216.9870 264.5995

358.1690

436.2220

480.2765

437.2242

457.2011

481.2801

508.3074691.3507522.2795 610.9991560.2029 648.4625 736.4348 755.4479783.1871

Elemental Composition Report

Single Mass Analysis (displaying only valid results)Tolerance = 10.0 PPM / DBE: min = -1.5, max = 50.0

Isotope cluster parameters: Separation = 1.0 Abundance = 1.0%

Monoisotopic Mass, Odd and Even Electron Ions92 formula(e) evaluated with 1 results within limits (up to 10 closest results for each mass)

Mass Calc. Mass mDa PPM DBE Score Formula

435.2184 435.2171 1.3 2.9 12.5 1 C27 H31 O5


Simvastatin Forced DegradationSimvastatin Forced Degradation

Simvastatin was degraded by five different methods:

—Acid Hydrolysis (Hydrochloric Acid)

—Base Hydrolysis (Sodium Hydroxide)

—Peroxide Oxidation (Hydrogen Peroxide)

—Temperature (Dry)

—Photo Degradation (Samples not yet run)Conditions for each method were modified to achieve approximately 10-20% degradation of the Simvastatin

UPLC analysis was carried out using a 7 minute gradient from 10mM Ammonium Acetate/25% Acetonitrile to 10mM Ammonium Acetate/90% Acetonitrile with Photodiode Array and Mass Spectrometric detection


Simvastation StandardSimvastation StandardBefore DegradationBefore Degradation

AU

0.00

0.20

0.40

0.60

0.80

1.00

1.20

Minutes3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00

AU

-0.015

-0.010

-0.005

0.000

Minutes3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00

PDA at 238nm

Simvastatin

Expanded ScaleAcetonitrileImpurity

Simvastatin

Simvastatin StandardBefore Degradation


Inte

nsi

ty

2.0x106

4.0x106

6.0x106

8.0x106

1.0x107

Minutes3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00

Inte

nsi

ty

0.0

5.0x107

1.0x108

1.5x108

Minutes3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00

Acid Hydrolysis of SimvastatinAcid Hydrolysis of Simvastatin

TIC Plot

Expanded Scale

Simvastatin

Simvastatin

60 Minute Acid HydrolysisOf Simvastatin

SimvastatinAcid

SimvastatinAcid


Inte

nsi

ty

2.0x106

4.0x106

6.0x106

8.0x106

1.0x107

Minutes3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00

Inte

nsi

ty

0.0

5.0x107

1.0x108

Minutes3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00

Base Hydrolysis of SimvastatinBase Hydrolysis of Simvastatin

TIC Plot

Expanded Scale

Simvastatin

Simvastatin

45 Minute Base HydrolysisOf Simvastatin

SimvastatinAcid

SimvastatinAcid


Peroxide Oxidation of SimvastatinPeroxide Oxidation of Simvastatin

Final conditions for Peroxide Degradation were determined to be 7.5% Hydrogen Peroxide for 45 minutes at 55° C

Degradation was carried out at a Simvastatin concentration of 1.0mg/ml

After 45 minutes of degradation, the standard solution was diluted 1 to 10 with Diluting Solution (~10mM Acetic Acid/80% Acetonitrile)

Degraded Simvastatin was injected at a concentration of 0.1mg/ml


Inte

nsi

ty

5.0x106

1.0x107

1.5x107

Minutes1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00

Inte

nsi

ty

0.0

5.0x107

1.0x108

Minutes1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00

Peroxide Oxidation of SimvastatinPeroxide Oxidation of Simvastatin

TIC Plot

Expanded Scale

Simvastatin

Simvastatin

45 Minute Peroxide OxidationOf Simvastatin


Inte

nsi

ty

0.0

5.0x106

1.0x107

1.5x107

2.0x107

Minutes1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00

Inte

nsi

ty

0.0

5.0x107

1.0x108

Minutes1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00

Temperature DegradationTemperature Degradationof Simvastatinof Simvastatin

TIC Plot

Expanded Scale

Simvastatin

Simvastatin

60 Minute TemperatureDegradation Of Simvastatin


ConclusionConclusion

UPLC® provides increased performance over traditional HPLC, speed, sensitivity, resolution

HPLC methods can be easily transferred to UPLC® using the ACQUITY® calculator

The extra efficiency of the ACQUITY UPLC® system requires a faster data capture rate detector 20-40Hz for reproducible quantitative analysis

Using the ACQUITY UPLC ® system with the column manager and the range of ACQUITY® BEH column chemistries method scouting can be rapidly accomplished using pH, temperature and stationary phases

Method development can be accomplished faster allowing better decisions to be made faster, improving productivity and quality


AcknowledgementsAcknowledgements

Mike Jones

Paul Rainville

Peter Alden


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