Chromatographic Advances for Pharmaceutical Analysisquimica.udea.edu.co/~carlopez/cromatohplc/hplc_devcelop_methods... · Chromatographic Advances for Pharmaceutical Analysis Robert

©2007 Waters Corporation

Chromatographic Advances for Chromatographic Advances for Pharmaceutical AnalysisPharmaceutical Analysis

Robert PlumbRobert PlumbWaters CorporationWaters Corporation

[email protected][email protected]

©2007 Waters Corporation

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Outline of PresentationOutline of Presentation

Why use higher temperatures

Theoretical considerations

Performance gains

Effect of temperature on analyte selectivity

Enabling viscous organic modifiers

Examples, natural products, metabolite ID

Column bleed at high temperatures

Effect on MS data capture rates

Conclusion


Ultra Performance LCUltra Performance LC™™exploiting small particle LCexploiting small particle LC


Sub 2Sub 2µµm Particle Chromatographym Particle ChromatographyUltraPerformance LCUltraPerformance LC®®

Efficiency inversely proportional to particle size

Flow rate increases with the inverse of particle size

Back pressure increases with the inverse square of particle size.

Thus sub 2µm columns bring :-

— Faster analysis faster flow ratefaster flow rate

— Higher operating pressures particle sizeparticle size


Why Use Higher TemperaturesWhy Use Higher Temperatures??

Reduce solvent viscosity = lower back-pressure—Facilitates the use of longer

columns—Allows smaller particles to be

used on conventional equipmentIncreased throughput—Lower back-pressure and

increased optimum flow rate means the flow can be increased

—analysis completed in a shorter time


Effect of Temperature on Effect of Temperature on Chromatographic PerformanceChromatographic Performance

Optimal Flow XBridge 2.1X100mm 3.5 um

0.0

2000.0

4000.0

6000.0

8000.0

10000.0

12000.0

14000.0

0 200 400 600 800 1000 1200

Flow Rate uL/min

USP

Pla

te C

ount

30 C

60 C

90 C

ACQUITY UPLC®


Performance GainsPerformance Gains

The efficiency of an isocratic chromatographic system is dependent upon:—Column length L

—Particle size 1/dp

—Mobile Phase flow rate

The peak capacity of a gradient chromatographic system is dependent upon—Column length

—Particle size

—Number of column volumes defining the gradient

Increasing column temperature does notdoes notincrease chromatographic efficiency


Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50

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

Isocratic Separations with UltraPerformance LCIsocratic Separations with UltraPerformance LC®®

effect of temperature effect of temperature

ACQUITY UPLCTM BEH C18 2.1 x 150 mm, 1.7 µmF = 0.4 mL/min T = 60 oC P = 7,300 PSI

N = 36,799


N = 35,643


N = 35,140


Effect of Temperature on PerformanceEffect of Temperature on Performancerapid gradients using 150mm columnrapid gradients using 150mm column

Average peak capacities(Peaks n=8) (Injections n=3)

350C: 283.4

650C: 284.8

950C: 275.8

Average peak capacities(Peaks n=8) (Injections n=3)

350C: 283.4

650C: 284.8

950C: 275.8

0.45mL/min

0.65mL/min

1.0mL/min

Elution of alkyl-phenones on a 2.1 x 150mm 1.7um column and an aqueous-organic gradient, maximum pressure 13,025psi


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

Increasing Efficiency HT UPLCIncreasing Efficiency HT UPLC®®

2.1 x 150 mm 1.7 2.1 x 150 mm 1.7 µµmm

Name Retention Time Area Height K Prime USP Resolution USP Tailing USP Plate Countthiourea 0.58 44198 62644 0.00 1.18 15865toluene 1.02 32307 36459 0.74 20.55 1.08 29565heptanophenone 1.46 49774 42285 1.15 16.32 1.05 34985octanophenone 1.77 48670 34903 2.04 9.00 1.03 36514amylbenzene 2.01 61932 38195 2.44 5.86 1.02 35165


N = 34,985


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


Linked Columns 2.1 x 300 mm 1.7 Linked Columns 2.1 x 300 mm 1.7 µµmm



N = 79,052


Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00


Linked Columns 2.1 x 450 mm 1.7 Linked Columns 2.1 x 450 mm 1.7 µµmm



N = 108,848


Effect of Temperature on Effect of Temperature on Analyte SelectivityAnalyte Selectivity

Raising the column temperature reduces analyte retention.

Temperature affects retention differently for different analytes.

This makes it difficult to transfer high temperature methods to preparative scale.

Simple method transfer from HPLC to high temperature, small particle LC or UPLC is not straightforward.


Effect of Temperature on Effect of Temperature on Analyte SelectivityAnalyte Selectivity

ACQUITY TUV ChA - ACQUITY TUV ChA 254nm

AU

0.00

0.05

0.10

0.15

0.20


AU

0.00

0.05

0.10

0.15

0.20


AU

0.00

0.05

0.10

0.15

0.20

Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

300C

600C

900C

1

5

4

32

3

6

2,31

2

1

5

5

7

74

6

47

6

10, 408 psi10, 408 psi

7,230 psi7,230 psi

5,281 psi5,281 psi

Separation pf 7 analgesics using an aqueous/organic gradient over 2 minutes using a 2.1 x 100mm 1.7μm C18 column on an ACQUITY UPLC® Chromatography System


Opening the Solvent EnvelopeOpening the Solvent Envelopeviscous organic modifiersviscous organic modifiers

Higher temperatures reduce mobile phase viscosity.

Reducing mobile phase viscosity allows the use of solvents too viscous for room temperature operation.

These solvents, such as ethanol, IPA, DMSO, DMF, have different selectivity from methanol and acetonitrile


Effect of Modifier on BackEffect of Modifier on Back--pressurepressure

15cm at 95C Pressure traces

psi

5000.00

5500.00

6000.00

6500.00

7000.00

7500.00

8000.00

8500.00

9000.00

9500.00

10000.00

10500.00

11000.00

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

ACN ~6000psi

IPA ~11,000psi

MeOH ~7800psi

Pressure traces for a 5-95% aqueous/organic gradients over 10 minute at a flow rate of 0.45mL/min


Reducing Analysis TimeReducing Analysis Timesharper peaks less retentionsharper peaks less retention


AU

0.00

0.20

0.40

0.60

0.80


AU

0.00

0.20

0.40

0.60


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

Isopropanol

Methanol

Acetonitrile

Gradient elution of alkyl-phenones over 10 minutes


Budesonide ImpuritiesBudesonide ImpuritiesIPA, sharper peaks, increased resolutionIPA, sharper peaks, increased resolution


AU

-0.02

0.00

0.02

0.04


AU

-0.02

0.00

0.02

0.04


AU

-0.02

0.00

0.02

0.04

Minutes2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

Isopropanol

Methanol

Acetonitrile


Improved ResolutionImproved Resolutionbudesonide impuritiesbudesonide impurities


AU

0.00

1.00

2.00

3.00

4.00


AU

0.00

1.00

2.00

3.00


AU

0.00

1.00

2.00

3.00

4.00

Minutes3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00

Isopropanol

Methanol

Acetonitrile


Natural Product AnalysisNatural Product Analysiswith high temperature UPLCwith high temperature UPLC®®

Natural products are often extremely complex mixtures

They present one of the most challenging tasks for separation science

Higher temperatures allow the use of longer columns and higher flow rates to produce very high resolution chromatograms

This combination allows the identification of new endogenous components


Longer SeparationLonger Separationpeak capacity >840peak capacity >840

16-Feb-2006 16667.00000000

Time5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00

%

0

100021606_PR_ESIPOS_007 1: TOF MS ES+

BPI2.38e3

15.51

11.41

0.43

0.46

5.002.95

1.09

2.08

6.00

6.97

7.87 8.73

11.42

14.74

11.51

11.53

15.52 33.94

28.86

27.58

16.25

27.57

20.54

16.77

18.51

21.42

25.8425.23

32.6631.17

33.97

47.1947.18

33.99

45.7641.66

16667.00000000

11.00 11.20 11.40 11.60 11.80 12.00

11.41

4

11.42

11.51

11.50

11.53

Ginseng extractGinseng extract


High Resolution Rapid Analysis High Resolution Rapid Analysis ginseng extractginseng extract

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

%

0


BPI3.49e3

6.444.08

2.20

2.01

1.80

1.59

1.341.331.10

2.37

3.07

2.54

3.00

3.13

3.92

3.26

3.43

6.42

6.30

4.20

4.81

4.30

4.99 6.23

5.91

5.88

5.09

5.69

5.46

7.806.70

7.61

7.32

6.91

7.88

8.338.38

Peak capacity = 500Peak capacity = 500

Peaks 1.2 seconds wide at base

Peaks 1.2 seconds wide at base

ACQUITY UPLC Q-Tof Premier™ system


Analyte Selectivity ChangesAnalyte Selectivity Changesginger root extractginger root extract


AU

0.00

0.05

0.10


AU

0.00

0.05

0.10


AU

0.00

0.05

0.10

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

Isopropanol

Methanol

Acetonitrile


Simavastatin Impurity ProfilingSimavastatin Impurity ProfilingQQ--ToFToF™™ AnalysisAnalysis


MS/MS Spectrum of SimvastatinMS/MS Spectrum of Simvastatin

O

OH+OH

CH3

CH3

OH+

OO

OOH

CH3 CH3 CH3

CH3

CH3 H

OH+

O

CH3

CH3

(1.0ppm)


Extracted ion Chromatogram 303Extracted ion Chromatogram 303


MS SpectrumMS SpectrumPeak at 6.9minPeak at 6.9min

O

OHO

O

OHCH3

CH3

CH3

CH3

CH3

H

OH

Simvastatin acid


MS/MS spectrumMS/MS spectrum

OH

OH+

OH

CH3

CH3

OH

O

OH+OH

CH3

CH3

OH+

O

CH3

CH3

O

OHO

O

OHCH3

CH3

CH3

CH3

CH3

H

OH


Metabolite IdentificationMetabolite Identification

17-Feb-2006 16667.00000000

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

%

0


BPI3.47e3

3.832.310.800.48

0.37

1.21

1.00

1.26 1.95

1.93

1.43

1.67

1.97

2.25

3.112.55

2.95 3.403.60

4.584.50

4.29

4.23

4.04

4.75

8.76

5.00 5.07

8.605.78

9.95

9.049.61 10.05


Acetominophen GlucuronideAcetominophen Glucuronide

17-Feb-2006 16667.00000000

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

%

0


328 0.50Da3.47e3

3.83

1.29

0.990.61

1.76

m/z200 400 600 800 1000

%0

100 6.05e3328.1041

295.1631 624.3478389.1334641.3907 804.3589


Effect of Narrow Peaks on MS Data Effect of Narrow Peaks on MS Data QualityQuality

Narrow peaks approximately 1 second wide at base require a fast data capture rate MS

10 – 20 spectra per second

How does this effect MS data quality and mass accuracy?


Issues with Accurate Mass and Fast Issues with Accurate Mass and Fast Separations Separations

To acquire a reliable accurate mass normally requires relatively long data acquisition times

UPLC peak widths are in the order of 1-2 seconds at the base

Accurate, reproducible peak integration requires >15 points across the peak

This means a duty cycle of 130mSec

Typically for Orbitrap acquisition times of 0.5 to 1Sec for high resolution and accurate mass

This would mean only 2-4 points across the peak


Number of Data Points Per Peak Number of Data Points Per Peak as recommended by as recommended by ““IonSource.comIonSource.com””

At least 15 to 20 points across a chromatographic peak for good quantification.

If you have fewer points you will not be able to describe the peak adequately and may lose information (e.g. peak top)

Reproducibility is negatively affected with fewer points and you will observe RSD’s increasing to unacceptable values.

http://www.ionsource.com/tutorial/msquan/tips.htm


Data dependent MS and MS/MS Data dependent MS and MS/MS

But what if you want to do MS/MS?—Well the then you need an even faster MS data

acquisition rate—Inter acquisition delay can become significantly

largeOption is not to acquire MS/MS in exact mass mode—This can lead to miss interpretation of the data—Fast switching can reduce MS/MS resolution

and accuracy—with a duty cycle of 350mSec, and UPLC®

there would be only 3 points across the peak total MS and MS/MS


Benefits of MSBenefits of MSEE for High Resolution for High Resolution LC/MSLC/MS

Data acquisition duty cycle <200mSec

—TOF 1 Scan 0.095mSec Delay 0.02mSec

—TOF 2 Scan 0.095mSec Delay 0.02mSec

—LockSpray every 11 scans

Overall Duty cycle 220mSec

For a 1 Sec peak 6 points across the peak for TOF 1 and 6 points across the peak in TOF 2.


Example DataExample Data

Time2.85 2.90 2.95 3.00

%

0

100

2.85 2.90 2.95 3.00

%

0

100PMD083_PR_101706_233 2: TOF MS ES+

BPI1.12e4

PMD083_PR_101706_233 1: TOF MS ES+ BPI

3.94e3


Dynamic Mass Resolution Dynamic Mass Resolution oaoa--Tof compared with Electrostatic FTTof compared with Electrostatic FT--ICR MSICR MS

Orbitrap: Resolution versus Cycle Time for different m/z values

0

10000

20000

3000040000

50000

60000

70000

80000

0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2

Cycle Time (sec)

Res

olut

ion

(FW

HM

)

400

800

1600

3200oa-TOF

2 spectra / sec

m/z


How Fast Can We Go Without How Fast Can We Go Without Loosing Mass Spectral Resolution?Loosing Mass Spectral Resolution?

m/z402 403

%

0

100 402.244

403.251

1s/scan

0.05s/scan

0.1s/scan

0.3s/scan

0.5s/scan

Spectra Overlay

Average Spectrum Resolution (m/z 402)= 20637

R = 20418

R = 21510

R = 19718

R = 21626

R = 19913


Rat Urine AnalysisRat Urine Analysishigh temperature high temperature –– high pressurehigh pressure

Pc 720 Pc 720 at baseat base

17-Feb-2006 16667.00000000

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

0

100021606_PR_ESIPOS_010

0.93

0.55

0.48

0.38

0.37

2.281.971.39

1.19 1.70

2.572.71

8.74

4.82

4.58

3.09

2.87

3.84

3.54

3.11

3.15

4.08

4.43

8.54

6.245.29

5.04 5.92

5.36

6.49 6.65

6.87 8.056.90 7.966.93

8.24

9.61

9.03

9.04

9.94

2.1 x 150mm ACQUITY UPLC2.1 x 150mm ACQUITY UPLC®® BEH BEH C18 column, operated at 90C18 column, operated at 90ººC, and C, and 900uL/min900uL/min


m/z160 180 200 220 240

%

0

100 1.74e4206.0458

170.0754 207.0880

Mass Accuracy with Very Fast UPLCMass Accuracy with Very Fast UPLC®®

m/z160 180 200 220 240

%

0

100 5.30e4190.0509

185.1614191.0893 231.1201

m/z160 180 200 220 240

%

0

100 3.83e4180.0667

221.0904m/z

200 250 300

%

0

100 2.01e4220.1182

246.2111309.2012

Kynurenic AcidKynurenic Acid2.3ppm2.3ppm

Pantothenic AcidPantothenic Acid1.4ppm1.4ppm

Hippuric AcidHippuric Acid3.3ppm3.3ppm

Xanthuric AcidXanthuric Acid2.4ppm2.4ppm


Column BleedColumn Bleedeffect of temperatureeffect of temperature

21-Feb-2006 16667.00000000

Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00

%

0

100022106_ESIPOS_PR_006 1: TOF MS ES+

550.627 0.50Da1.85e3

2.682.542.48

2.452.36

2.26

2.05

2.72

022106_ESIPOS_PR_006 1: TOF MS ES+ 522.597 0.50Da

8162.722.672.49

2.402.34

2.212.18

2.06

2.74


3.22e32.21

2.18

2.182.05

1.951.84

2.23 2.482.49 2.682.75


5.72e32.232.202.06

1.96

1.841.72

2.542.53 2.63 2.77


2.54e32.21

2.182.05

2.042.021.83

1.70

2.232.26

2.322.41

2.55 2.72

XIC Silica C18 1.8 μm 2.1 x 50 mm at 90 °C


Column BleedColumn Bleedeffect of temperatureeffect of temperature

01285603310C02 2_1 x 100 mm 90 C 1 MeCN01285603310C02 2_1 x 100 mm 90 C 1 MeCN

Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50

%

0

100

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50

%

0

100020806_ESIPOS_PR_044 1: TOF MS ES+

550.622 0.50Da5.40e3

2.59

2.55

2.35


1.87e31.91

1.89


1.23e31.471.44

1.40


2.31e32.99

1.681.75

2.082.18

3.06


5813.00

1.931.791.73

1.48 2.09 2.193.04

ACQUITY BEH C18 Column


ConclusionConclusion

High temperature LC and UPLC® allow faster separation

Efficiency is not improved at higher temperatures

Higher column temperature need higher flow rates

Higher temperatures enable the use of longer columns

Alternative solvents can be used at higher temperatures for unique selectivity

Column bleed can be an issue at higher temperatures—ACQUITY® BEH offers the minimal column bleed.

Narrow peaks require fast scanning UV and MS detectors


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Chromatographic Advances for Pharmaceutical Analysisquimica.udea.edu.co/~carlopez/cromatohplc/hplc_devcelop_methods... · Chromatographic Advances for Pharmaceutical Analysis Robert

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