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©2012 Waters Corporation 1

Transcript of ©2012 Waters Corporation 1 · Ir(fppy)3 N F F N AU 0.06 N F F Ir F F N F Ir(fppy)3 Isomer...

Page 1: ©2012 Waters Corporation 1 · Ir(fppy)3 N F F N AU 0.06 N F F Ir F F N F Ir(fppy)3 Isomer Ir(fppy)3 -F Ir(fppy)3-2F 1.935 6 0.03 N F F F Ir N F N F Ir N F F N F F I (f )3 F Ir N

©2012 Waters Corporation 1

Page 2: ©2012 Waters Corporation 1 · Ir(fppy)3 N F F N AU 0.06 N F F Ir F F N F Ir(fppy)3 Isomer Ir(fppy)3 -F Ir(fppy)3-2F 1.935 6 0.03 N F F F Ir N F N F Ir N F F N F F I (f )3 F Ir N

©2012 Waters Corporation 2

Page 3: ©2012 Waters Corporation 1 · Ir(fppy)3 N F F N AU 0.06 N F F Ir F F N F Ir(fppy)3 Isomer Ir(fppy)3 -F Ir(fppy)3-2F 1.935 6 0.03 N F F F Ir N F N F Ir N F F N F F I (f )3 F Ir N

©2012 Waters Corporation 3

Page 4: ©2012 Waters Corporation 1 · Ir(fppy)3 N F F N AU 0.06 N F F Ir F F N F Ir(fppy)3 Isomer Ir(fppy)3 -F Ir(fppy)3-2F 1.935 6 0.03 N F F F Ir N F N F Ir N F F N F F I (f )3 F Ir N

©2012 Waters Corporation 4

Page 5: ©2012 Waters Corporation 1 · Ir(fppy)3 N F F N AU 0.06 N F F Ir F F N F Ir(fppy)3 Isomer Ir(fppy)3 -F Ir(fppy)3-2F 1.935 6 0.03 N F F F Ir N F N F Ir N F F N F F I (f )3 F Ir N

©2012 Waters Corporation 5

Page 6: ©2012 Waters Corporation 1 · Ir(fppy)3 N F F N AU 0.06 N F F Ir F F N F Ir(fppy)3 Isomer Ir(fppy)3 -F Ir(fppy)3-2F 1.935 6 0.03 N F F F Ir N F N F Ir N F F N F F I (f )3 F Ir N

Waters UPC² Workshop Series

Applications

©2012 Waters Corporation 6

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Lipids

©2012 Waters Corporation 7

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UPC²/MSUPC²/MSPolar Lipids Mixture Rapid Screening Polar Lipids Mixture Rapid Screening Separation (Brain, porcine standards)Separation (Brain, porcine standards)Separation (Brain, porcine standards)Separation (Brain, porcine standards)

1.6x108

PC

1.2x108

Inte

nsity

8.0x107

LPC

SM

4.0x107

CERPG PE LPE

00

©2012 Waters Corporation 8

0.0

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

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UPC² and LipidomicsUPC² and LipidomicsEvaluation Mouse Heart ExtractEvaluation Mouse Heart Extract

Glycerolipids(NH + adducts) PC

2.0x108

(NH4+ adducts) PC

ty

1.5x108

Inte

nsi

5.0x107

1.0x108

PE

SM

Cardiolipins

TAG

DAG

MAG

LPC

0.0

0 00 1 20 2 40 3 60 4 80 6 00 7 20 8 40 9 60 10 80 12 00

LP

E

PGPS

©2012 Waters Corporation 9

Minutes0.00 1.20 2.40 3.60 4.80 6.00 7.20 8.40 9.60 10.80 12.00

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UPC² Polar Lipids Mixture UPC² Polar Lipids Mixture MS ESI+ SIR OverlayMS ESI+ SIR Overlayyy

1.2x108

496.3

= 4

96.3

9.0x107

LP

Cm

/z =

4

PC

m/z

=

Inte

nsity

6.0x107

4.2

82.2

= 6

48.5

2.4 731.5

813.6

6.4 815.6

3.0x107P

Em

/z =

464

PE

m/z

= 4

8

CE

Rm

/z =

PE

m/z

= 7

02

SM

m

/z =

S

M m

/z =

PG

m/z

= 7

66

SM

m

/z =

0.0

L LPP

©2012 Waters Corporation 10

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50

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Vitamins

©2012 Waters Corporation 11

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Vitamin E OTC FormulationsVitamin E OTC Formulations

2.74

1

1.00

1.20

1.40

1.60

3.19

3

3.37

7

3.57

0

AU

0.00

0.20

0.40

0.60

0.80

Peak 1 α-TocopherolPeak 2 β-TocopherolPeak 3 γ-Tocopherol

2.74

6

6AU

0.80

1.00

1.20

1.40Peak 4 δ-Tocopherol

3.19

7

3.37

6

3.57

1

0.00

0.20

0.40

0.60

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

Instrument ACQUITY UPSFC, PDA (293 nm), EMPOWER 3Column HSS C18 SB (3.0 100 mm, 1.8 µm)

Run time 5 minutesMobile phase Gradient 0.5% MeOH to 5% over 5 minutes

Fl t 2 5 L/ i

©2012 Waters Corporation 12

Flow rate 2.5 mL/minPressure 130 bar (1885 psi)

Temp 50 CInjection 1.0 µl injectionSample Contents of Vit E capsule in Hexane

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Vitamin Vitamin E E Degradation Analysis Degradation Analysis g yg y

2.74

1

2.83

0

0.009

0.010

0.011

1.554 Peak 1

287.2339.4 371.4

AU

0.000

0.002

0.004

1.648 Peak 2

2.94

4

AU

0 002

0.003

0.004

0.005

0.006

0.007

0.008

282.4 367.8 387.0

AU

0.000

0.002

2.598 Peak 3229.2

268.2331 2

AU0.010

0.020

1.55

4

1.64

7 2.59

8

-0.003

-0.002

-0.001

0.000

0.001

0.002

Minutes1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00

331.20.000

2.742 Peak 4212.7

293.1

AU

0.00

2.00

2.829 Peak 5

293.1352.2 366.6

AU

0.00

0.02

0.04

2.943 Peak 6

AU 0 02

0.04

©2012 Waters Corporation 13

293.1

A

0.00

0.02

nm220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 400.00

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Vitamin A formsVitamin A forms

tate

- 1.20

Ret

inyl

Ace

t

0.90

1.00

1.10

0 60

0.70

0.80

tate

- 3.

354

Retin

ol -

3.82

6

AU

0.40

0.50

0.60

207

04 8Re

tinol

Pal

mit R

0.10

0.20

0.30

©2012 Waters Corporation 14

1. 1.3

1.79

71.

938

2.44

0

3.21

8

4.64

4

5.80

7

6.37

6

0.00

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 7.50 8.00

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Separation Vitamin A forms (Zoom)Separation Vitamin A forms (Zoom)

0.060

p ( )p ( )

1.20

7

0.045

0.050

0.0551.

304

0 030

0.035

0.040

5 6

AU

0.020

0.025

0.030

97 0.41

1

6 43

1.15

5

1.51

686 1.

797

.938

3 2.44

0

86 6

3.21

826

8

55 4 1 493

.644

07 0 6.37

6

3

0.005

0.010

0.015

©2012 Waters Corporation 15

0.29

0.79

6

0.94 1.58 1

2.15

3

2.88

3.02

6

3.2

4.05

4.14

4

4.39

14.

4 44.

701

4.93

75.

064

5.45

8

5.67

95.

80

6.01

0

6.17

7

6.69

3

0.000

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50

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Fat Soluble Vitamin MixturesFat Soluble Vitamin Mixtures

0.060

0.040

0.050

AU 0.030

0.010

0.020

0.000

Mi t0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

©2012 Waters Corporation 16

Minutes

Red - MS TIC (ESi+)Blue - UV at 317 nmBlack - UV at 262 nm

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OLEDs

©2012 Waters Corporation 17

Page 18: ©2012 Waters Corporation 1 · Ir(fppy)3 N F F N AU 0.06 N F F Ir F F N F Ir(fppy)3 Isomer Ir(fppy)3 -F Ir(fppy)3-2F 1.935 6 0.03 N F F F Ir N F N F Ir N F F N F F I (f )3 F Ir N

OLED Separation and Impurity OLED Separation and Impurity ProfilingProfiling

Organic Light Emitting Diode (OLED)

gg

(Cathode)

( d )(Anode)

(Glass board)

©2012 Waters Corporation 18

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OLED Degradation Investigation OLED Degradation Investigation with LC/UV/MS with LC/UV/MS

FlrpicStructural Structural Isomers

Flrpic

α-NHP

©2012 Waters Corporation 19

TCTABAlq

Normal Phase LC Separation

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UPC² Organic Light Emitting Diodes UPC² Organic Light Emitting Diodes OLEDsOLEDs

Ir(fppy)3Area = 0.4%Rt = 0.1%

α NHP

FF

1.00

9

1.42

10.075

0.100

TCTAArea = 1.1%Rt = 0.1% Balq (a)

Area = 1 1%

α-NHPArea = 0.4%Rt = 0.1%

N

N

FIr

N

F

1.92

0

2.02

8

2.47

1

AU

0.050

Area = 1.1%Rt = 0.1%

N

F F

0.025

0.000

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

©2012 Waters Corporation 20

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UPC² OLEDsUPC² OLEDsIrIr((fppyfppy))33 Degraded SampleDegraded Sample(( ppyppy))33 g pg p

0.12

1.019 Peak 1

258.4

1.01

9

0.09

225.4

0.054

0.072

AU

0.06

AU

0.036

0.03

373.4

0.018

nm220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 400.00

0.00

©2012 Waters Corporation 21

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

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UPC²/MS OLEDsUPC²/MS OLEDsIrIr((fppyfppy))33 Degraded SampleDegraded Sample(( ppyppy))33 g pg p

03

9.0x1071.004 Peak 1 - SQ 1: MS Scan 1: 300.00-1000.00 ES+, Centroid, CV=Tune

763.92

2.8x107

1.00

7.2x107

761.95

ensi

ty

2.1x107

nten

sity

5.4x107

Inte

7.0x106

1.4x107

In

3.6x1070.0

m/z744.80 748.60 752.40 756.20 760.00 763.80 767.60 771.40 775.20 779.00

1.8x107

©2012 Waters Corporation 22

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

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Standards and SpectraStandards and SpectraFlrpicFlrpic and MS ES+ m/z valuesand MS ES+ m/z valuespp

1.345 Peak 2 - SQ 1: MS Scan 1: 300.00-1000.00 ES+, Centroid, CV=Tune

743.93

745.90

Inte

nsity

1.8x106

2.7x106

3.6x106

1.446 Peak 3 - SQ 1: MS Scan 1: 300.00-1000.00 ES+, Centroid, CV=Tune

743.97

745.91

Inte

nsity

2.4x106

3.6x106

4.8x106

1.601 Peak 4 - SQ 1: MS Scan 1: 300.00-1000.00 ES+, Centroid, CV=Tune

743.90

745.90

746.97

Inte

nsity

2x106

3x106

4x106

m/z=745.9 m/z=745.9 m/z=763.9

ES+

20

9.0x107

0.0

9.0x105

m/z734.40 737.10 739.80 742.50 745.20 747.90 750.60 753.30 756.00 758.70

0.0

1.2x106

m/z738.00 740.00 742.00 744.00 746.00 748.00 750.00 752.00 754.00

0

1x106

m/z739.20 741.30 743.40 745.50 747.60 749.70 751.80 753.90 756.00

1.922 Peak 6 - SQ 1: MS Scan 1: 300.00-1000.00 ES+, Centroid, CV=Tune763.93

2.8x107

2.731 Peak 7 - SQ 1: MS Scan 1: 300.00-1000.00 ES+, Centroid, CV=Tune745.95

2.4x107

1.004 Peak 1 - SQ 1: MS Scan 1: 300.00-1000.00 ES+, Centroid, CV=Tune763.92

2.8x107

1.00

3 1.92

2.73

0

7.2x107

761.97

765.08

Inte

nsity

0.0

7.0x106

1.4x107

2.1x107

m/z757.80 759.60 761.40 763.20 765.00 766.80 768.60 770.40 772.20

743.96

747.05

Inte

nsity

0.0

6.0x106

1.2x107

1.8x107

m/z738.00 740.00 742.00 744.00 746.00 748.00 750.00 752.00 754.00 756.00

3.605 Peak 8 - SQ 1: MS Scan 1: 300.00-1000.00 ES+, Centroid, CV=Tune2 94

4.101 Peak 9 - SQ 1: MS Scan 1: 300.00-1000.00 ES+, Centroid, CV=Tune

761.95

765.01

Inte

nsity

0.0

7.0x106

1.4x107

2.1x107

m/z759.60 760.80 762.00 763.20 764.40 765.60 766.80 768.00 769.20

m/z=763.9 m/z=763.9 m/z=746.0 m/z=727.9 m/z=806.0

.604

Inte

nsity

3.6x107

5.4x107725.97

727.94

729.01

Inte

nsity

0.0

1.6x106

3.2x106

4.8x106

6.4x106

m/z722.40 723.80 725.20 726.60 728.00 729.40 730.80 732.20 733.60 735.00

804.01

805.99

806.96

Inte

nsity

0.0

1.2x106

2.4x106

3.6x106

4.8x106

m/z802.80 803.70 804.60 805.50 806.40 807.30 808.20 809.10 810.00

1.34

5

1.44

8

1.60

01.

643

3.

4.10

2

1.8x107

3.6x10

©2012 Waters Corporation 23

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

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UPC²/MS OLEDsUPC²/MS OLEDsIrIr((fppyfppy)3 Degraded Sample)3 Degraded Sample(( ppyppy) g p) g p

0.12

1.01

9

0.09

Ir(fppy)3

N

FF

N

AU

0.06

N

F

F

Ir

F

F

N

F

Ir(fppy)3Isomer

Ir(fppy)3 -2FIr(fppy)3 -F1.

935

6

0.03

N

F

F

Ir

N

FN

F

F

N

F

Ir

N

F

F

N

F

F

I (f )3 F

Ir

N

F

N

F

1.36

0

1.46

3

1.61

9

2.74

6

3.62

2

4.12

4

0.00

Ir(fppy)3–FIosmers

N

F F

©2012 Waters Corporation 24

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

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UPC²/MS OLEDsUPC²/MS OLEDsIrIr((fppyfppy))33 Degraded SampleDegraded Sample(( ppyppy))33 g pg p

10 X Reduction in runtime over traditional NPLC method

4 major constituents separated

Seven impurities identified

NP was 70-minute run with co-elutions NP was 70-minute run with co-elutions

©2012 Waters Corporation 25

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USP Method Conversions

©2012 Waters Corporation 26

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UPCUPC22 vs. Normal Phase vs. Normal Phase HPLCHPLC

©2012 Waters Corporation 27

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Normal Phase Method Conversion Normal Phase Method Conversion EstradiolEstradiol to UPCto UPC2™2™

30.8

56

0 .0014

0.0016

0.0018

0.0020

Normal Phase HPLCCost per run ~ $5.89

Compound RT %Area S/N Unk Impurity 6 24 0 006 2 9

35.8

19

AU

0 .0004

0.0006

0.0008

0.0010

0.0012

Unk. Impurity 6.24 0.006 2.9Unk. Impurity Not Found --- --- Unk. Impurity 10.86 0.01 2.7Unk. Impurity Not Found --- --- Unk. Impurity 20.85 0.018 3Unk. Impurity 26.63 0.021 3.2Estradiol 30.86 99.87 ---

6.23

7

10.8

55

20.8

50

26.6

32

-0.0004

-0.0002

0.0000

0.0002

M inutes0.0 0 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00

Main Impurity 36.81 0.077 9.2

Compound RT %Area S/N

ACQUITY UPC2

Cost per run ~ $0.05

pUnk. Impurity 2.26 0.012 3.4Unk. Impurity 2.59 0.004 1.9Unk. Impurity 3.34 0.01 3.1Unk. Impurity 5.66 0.006 1.7Unk. Impurity 6.15 0.016 5.5Unk. Impurity 8.13 0.013 3.1

©2012 Waters Corporation 28

p yEstradiol 8.81 99.89 ---Main Impurity 9.99 0.046 16

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Chiral Separations

©2012 Waters Corporation 29

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Chiral Comparison of Chiral Comparison of ACQUITYACQUITY UPCUPC22

vs. Normal Phase vs. Normal Phase HPLCHPLC

©2012 Waters Corporation 30

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UPC² Enantiomeric Excess UPC² Enantiomeric Excess Determination of Benzyl Mandelate Determination of Benzyl Mandelate yy

R R 7 28

SFC chromatogram of R- and S- benzyl mandelate at 0.20 mg/mL of each enantiomer.

AU 0.20

R S RS=7.28

0.00

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

SFC h t f R b l d l t t 2 / L

0.004 RS

SFC chromatogram of R-benzyl mandelate at 2 mg/mL.

0.02% impurity

AU

0.000

0.002

©2012 Waters Corporation 31

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

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UPCUPC2 2 SubSub--2µm 2µm ChiralChiral SeparationsSeparationsWarfarinWarfarin

2.21

8

2.32

0

0.20

0.30 Prototype #1 1.7 µm 2.1 X 50 mm

2

AU

0.00

0.10

AU

0.20

0.30 Commercially Available 5.0 µm 2.1 X 150 mm

2.88

7

3.37

7

0.00

0.10

2

0.30 Prototype #2 1.7 µm 2.1 X 50 mm

0.96

21.

041

AU

0.10

0.20

Prototype #2 1.7 µm 2.1 X 50 mm

©2012 Waters Corporation 32

0.00

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

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Methamphetamine

©2012 Waters Corporation 33

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MethamphetamineMethamphetamine

Many of the illicit drugs occur as optical isomers with different psychotropic activities For example

pp

activities. For example, methamphetamine is a widely abused chiral drug.

The S form has 5 times the psycho-stimulant effect than that of the R form

(S)HN (R)HN

S(+)-methamphetamine R(-)-methamphetamine

stimulant effect than that of the R form. Methamphetamine is easy to make and

its precursors are readily available in the form of over-the-counter (OTC) drugs.

The impurity profile and enantiomeric The impurity profile and enantiomeric ratio of methamphetamine can provide valuable information to law enforcement agencies regarding the precursors as well as the synthetic routes. well as the synthetic routes.

Current analytical methods, including GC and CE, often involve long run times or time-consuming sample work-up steps.

©2012 Waters Corporation 34

p

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Optimized Chiral Separation of R, SOptimized Chiral Separation of R, S--methamphetaminemethamphetaminepp

Chiral separation was achieved in less than 5 min.less than 5 min.

This represents a significant improvement in speed of analysis compared to GC or CE.U

0.004 1 2Rs=1.84

A

0.000

Minutes0.00 1.00 2.00 3.00 4.00

Optimized SFC chromatogram of methamphetamine. 1: S-MA; 2: R-MA.

©2012 Waters Corporation 35

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Separation of methamphetamine with Separation of methamphetamine with its precursors: the fingerprintits precursors: the fingerprintp g pp g p

The enantiomeric ratio of R/S methamphetamine and their methamphetamine and their precursors are valuable information for law enforcement agencies to determine

AU

0.002 1 2

3

4

geographical location and manufacturing sophistication.

Simultaneous separation of R, S-methamphetamine and their two

0.000

3

methamphetamine and their two common precursors in 6 min.

SFC UV chromatogram of a mixture of: 1: S

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00

SFC UV chromatogram of a mixture of: 1: S-MA; 2: R-MA ; 3: (1S,2S)-pseudoephedrine; and 4: (1R, 2S)-ephedrine.

©2012 Waters Corporation 36

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Drug TestingDrug Testingg gg g

1000000S-MA

Meth. is commonly tested during workplace drug

600000

800000

ak A

rea

R-MA

Linear (S-MA)

Linear (R-MA)

during workplace drug screenings.

Laboratories are required to detect methamphetamine at

R² (S) = 0.998R² (R) = 0.998

0

200000

400000

Pea

40% of its confirmatory level of 250 ng/mL.

The calibration curves of R- and S-methamphetamine ranging 0

0 200 400 600 800 1000

Concentration (ng/mL)

S-methamphetamine ranging from 50 ng/mL to 1,000 ng/mL, using SFC APCI MS.

Calibration curves of R- and S-methamphetamine obtained using SFC APCI MS.

©2012 Waters Corporation 37

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UPCUPC2 2 Drugs of Abuse Drugs of Abuse MethamphetamineMethamphetamine

CE/MS

15-20 min

pp

15 20 min

GC/MSS/N=11.6 S/N=10.2

UPC2/MS/MS (ESI) 6 min

10-12 min

with derivatization step

S/ 6 /

LC/MS/MS

40-60 min 5 ng/mL for each enantiomer (25 pg on column)

©2012 Waters Corporation 38

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Simultaneous separation of Simultaneous separation of methamphetamine and amphetaminemethamphetamine and amphetaminep pp p

Since amphetamine is the main metabolite of methamphetamine metabolite of methamphetamine, to test positive for meth., amphetamine also has to be detected in the sample.

0.003

1

2

34

Simultaneous chiral separation of amphetamine and methamphetamine in less than 5 minutes

AU

minutes. 0.000

Minutes0.00 1.00 2.00 3.00 4.00 5.00

SFC UV chromatogram of a mixture of amphetamine and methamphetamine. 1: S-MA; 2: R-MA ; 3: S-AMP; and 4: R-AMP.

Minutes

©2012 Waters Corporation 39

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Paracetamol

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ParacetamolParacetamol and Process Impuritiesand Process Impuritiespp

A mixture of paracetamol is analysed by UPC² and UPLC.

Sample Composition:

S1 S2

Code  Compounds  Mw(g.mol‐1) me1 (mg) Vtot (ml) EtOH Vµl S1 Vtot (ml) mg/ml

A 4 hydroxyacetophenone 136 50 10 20 10 0.010

B p‐nitrophenol 139 50 10 20 10 0.010

C 2 hydroxyacetophenone 136 50 10 20 10 0.010

D 4 chloroacetanilide 169.6 50 10 20 10 0.010

E acetanilide 135 50 10 20 10 0.010

F phenacetin 179 50 10 20 10 0.010

G 3 aminophenol 151 50 10 20 10 0 010G 3 aminophenol 151 50 10 20 10 0.010

H 4 aminophenol (paracetamol) 151 100 10 60 10 0.060

I 2 aminophenol  151 50 10 20 10 0.01

S2 i H O f UPLC d i H f UPC²

©2012 Waters Corporation 41

S2 in H2O for UPLC and in Heptane for UPC²

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Method ParametersMethod Parameters

SFC UPLC UPC²

System: H-CLassColumn: BEH RP18 2.1x50mm

1.7µm

System: UPC²Column: BEH 3.0x100mm

1 7 1.7µm

T°: 55°CF: 0.9 ml.min-1

Solvent A: Water 10mM acetate mmoni m pH 5

1.7µm T°: 40°C

F: 2ml.min-1

Solvent : Methanolammonium pH:5Solvent B: Acetonitrile

Vinj: 1µL

So e t et a oVinj: 2µl

time (min) % CO2 % MeOH Flow (ml.min‐1)

0 98 2

time (min) % A % B Flow (ml.min‐1)

RUN

0 100 0 0.9

3 73 27 0 9

RUN 

0 98 2 2

0.4 98 2 2

2 93 7 2

2.4 93 7 2

©2012 Waters Corporation 42

RUN  3 73 27 0.9

3.5 100 27 0.9

2.6 80 20 2

2.7 98 2 2

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UPLC Results @ 260nm UPLC Results @ 260nm @@

0.14 H

0.10

0.12

AU

0.08

0 0

A

0.04

0.06

0.00

0.02 G DCB

FEI

©2012 Waters Corporation 43

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

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UPC² Results @ 220nm UPC² Results @ 220nm

HH

A

C

B

C

DEFG

I

©2012 Waters Corporation 44

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UPC² Results @ 260nm UPC² Results @ 260nm

H

A

C

B

C

DEFG

I

©2012 Waters Corporation 45

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UPC² UPC² vsvs UPLCUPLC

H

H

A

CG

B D E FG

I

0.10

0.12

0.14H

A

AU

0.04

0.06

0.08

G DC

BF

A

EI

©2012 Waters Corporation 46

0.00

0.02

0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00

G CFEI

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Analgeasics

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AnalgesicsAnalgesicsgg

A mixture of analgesics are analysed by UPC² and UPLC.

Sample Composition

S1 S2

Code  Compounds  Mw(g.mol‐1) me1 (mg) Vtot (ml) EtOH Vµl S1 Vtot (ml) heptane mg/ml

1 acetanilide 135 10 10 500 20 0.050

2 caffeine 194 10 10 1000 20 0.100

3 acethylsalicylic 180 10 10 1000 20 0.100

4 3 aminophenol 151 50 10 500 20 0.250

5 phenacetin 179 50 10 500 20 0.250

6 4 aminophenol (paracetamol) 151 100 10 250 20 0.250

7 2 aminophenol 151 50 10 500 20 0 2507 2 aminophenol 151 50 10 500 20 0.250

8 Acide salicylic 138 10 10 1000 20 0.100

©2012 Waters Corporation 48

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Method ParametersMethod Parameters

SFC UPLC UPC²

System: H-CLassColumn: BEH RP18 2.1x50mm 1.7µm

T°: 60°C

System: UPC²Column: BEH 2 ep 3.0x100mm

1 7µm T : 60 CF: 0.9 ml.min-1

Solvent A: Water 10mM ammonium formate pH:3

Solvent B: MeOH

1.7µm T°: 40°C

F: 2ml.min-1

Solvent : MethanolVinj: 32µlSolvent B: MeOH

Vinj: 1µLVinj: 32µl

time (min) % CO2 % MeOH Flow (ml.min‐1)

0 99 1time (min) % A % B Flow (ml.min‐1)

RUN 

0 100 0 0.6

3 64 36 0.6

RUN 

0 99 1 2

0.5 99 1 2

3.5 60 40 2

4.0 60 40 2

©2012 Waters Corporation 49

3.5 100 0 0.64.1 99 1 2

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UPLC Results 280nmUPLC Results 280nm

20

0.35

Caf

fein

e -1

.32

0.25

0.30

-0.7

57

heno

l -0.

958

phen

ol -

1.06

3

90 558 -1

.735

76

AU

0.15

0.20

ceta

min

ophe

n -

2-A

ceta

mid

oph

3-A

ceta

mid

op

ceta

nilid

e -1

.3

licyl

ic a

cid

-1.5

tyls

alic

ylic

aci

d

enac

etin

-2.

27

0.05

0.10

Ac Ac Sal

Ace

t

Ph

0.00

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

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UPC² UPC² ResultsResults @ 220nm Vinj: 2µL@ 220nm Vinj: 2µL@ j µ@ j µ

6

52

3

57

2

1

3

4

8

©2012 Waters Corporation 51

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UPLC vs UPC²UPLC vs UPC²

0 25

0.30

0.352 UPLC

AU

0 10

0.15

0.20

0.25

0.00

0.05

0.10

Mi t0.00 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

6 7 41 8 3 5

Minutes

5

6

7

0.70

20.60

0.50

UPC²

1

3

4

0.40

0.30

0.20

©2012 Waters Corporation 52

4

8

0.00 1.00 2.00 3.00 4.00

0.00

0.10

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Polymer Additives

©2012 Waters Corporation 53

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Polymer AdditivesPolymer Additivesyy

Lowinox 44B25

Irganox 1010

Tinuvin 328BHTIrganox 1330

©2012 Waters Corporation 54

Tinuvin P Tinuvin 312

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UPC² ConditionsUPC² Conditions

Conditions:– Cosolvent: MeOH/MeCN 1/1Cosolvent: MeOH/MeCN 1/1

– Column: BEH 2EP 3.0x100mm 1.7µm

– Gradient: 1% for 1min, then ramp to 20% over 2.5min, 20% for 30s then re-equilibrationthen re equilibration

– Flow: 2.0mL/min

– Temp: 60°CP 120 b– Pressure: 120 bars

– Diluent: Heptane/Ethanol 9/1

– Concentration: 0.2mg/mL of each

– Detection: UV at 220nm

©2012 Waters Corporation 55

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UPC² ResultsUPC² Results

5 min cycle time

836

ox 1

330

- 2.8

41

3.73

3

0.30

0.32

0.34

0.36

0.38

BH

T - 0

.407

Tinu

vin

P -

0.95

1

vin

328

- 1.2

25

Tinu

vin

312

- 1.8

gaar

d 44

5 - 2

.698

Irgan

o

351

Low

inox

44B

25 -

0.20

0.22

0.24

0.26

0.28

Tinu

v

Nau

Irgan

ox 1

010

- 3.3

AU

0.10

0.12

0.14

0.16

0.18

-0 02

0.00

0.02

0.04

0.06

0.08

©2012 Waters Corporation 56

-0.04

0.02

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 3.80 4.00 4.20 4.40 4.60 4.80 5.00

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FasterFaster GradientGradient

Faster gradient test – 3.5min cycle time

0.38

0.40

0.42

0.44

0.46

0.48

0.26

0.28

0.30

0.32

0.34

0.36

AU

0.14

0.16

0.18

0.20

0.22

0.24

0.02

0.04

0.06

0.08

0.10

0.12

©2012 Waters Corporation 57

-0.04

-0.02

0.00

Minutes0.00 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 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50

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UPLC Conditions UPLC Conditions

ACQUITY BEH C18; 2.1 mm x 50 mm (60°C)

Flow rate: 1 ml/min

Gradient separation, water to acetonitrile

From 48% to 100% B in 2 minutes; 1 minute at 100% B and From 48% to 100% B in 2 minutes; 1 minute at 100% B and back to initial

0.5µl injection (0.25 mg/ml of each additive, @220 nm)

©2012 Waters Corporation 58

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UPLC Results UPLC Results Polymer Additives (Industrial Grade)Mixture With 8 Compounds

uvin

P

328

30

0.90

1.00Ti

nuvi

n31

2

4B25

445

Tinu

vin

3

Irgan

ox13

3

U

0.60

0.70

0.80

in31

2

Tinu

v

in31

2

BHT Lu

win

ox44

Nau

gard

4

gano

x10

10

AU

0.30

0.40

0.50

Imp

1 Ti

nuv

Imp

2 Ti

nuvi

Impu

rity

c

Irg0 00

0.10

0.20

©2012 Waters Corporation 59

0.00

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00

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UPLC Results UPLC Results

Polymer Additives (Industrial Grade)Mixture With 11 Compounds

vin

P

1081

x13

30

0.80

0.90Ti

nuv

inuv

in 3

12

x 44

B25

Irgan

ox

gard

445

Tinu

vin

328

10Irg

anox

168

U

0.50

0.60

0.70

p 1

Tinu

vin

312 Ti

purit

y a

2 Ti

nuvi

n 31

2

BHT

Luw

inox

Nau

g

purit

y c

Irgan

ox 1

01

Imp

1 Irg

afos

rg

anox

107

6Irg

afos

168

AU

0.20

0.30

0.40

Imp

Imp

Imp

Imp Ir

0.00

0.10

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00

©2012 Waters Corporation 60

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UPC² vs UPLC UPC² vs UPLC 0.

407

P -

0.95

1

225

vin

312

- 1.8

36

2.69

8

Irgan

ox 1

330

- 2.8

41

nox

44B

25 -

3.73

3

0 2 4

0 . 2 6

0 . 2 8

0 . 3 0

0 . 3 2

0 . 3 4

0 . 3 6

0 . 3 8

2 4

6

81B

HT

- 0

Tinu

vin

Tinu

vin

328

- 1.2

Tinu

v

Nau

gaar

d 44

5 -

Irgan

ox 1

010

- 3.3

51

Low

in

AU

0 . 0 6

0 . 0 8

0 . 1 0

0 . 1 2

0 . 1 4

0 . 1 6

0 . 1 8

0 . 2 0

0 . 2 2

0 . 2 4 23

45

7

1

- 0 . 0 4

- 0 . 0 2

0 . 0 0

0 . 0 2

0 . 0 4

M in u t e s0 . 0 0 0 . 2 0 0 . 4 0 0 . 6 0 0 . 8 0 1 . 0 0 1 . 2 0 1 . 4 0 1 . 6 0 1 . 8 0 2 . 0 0 2 . 2 0 2 . 4 0 2 . 6 0 2 . 8 0 3 . 0 0 3 . 2 0 3 . 4 0 3 . 6 0 3 . 8 0 4 . 0 0 4 . 2 0 4 . 4 0 4 . 6 0 4 . 8 0 5 . 0 0

1 00 2

U 0 60

0.70

0.80

0.90

1.00 2

3

4

6

1 Ti

nuvi

n31

2

2 Ti

nuvi

n31

2

1

urity

c

AU

0.20

0.30

0.40

0.50

0.60

5 78

©2012 Waters Corporation 61

Imp

Imp

Impu

0.00

0.10

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00

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HAP

©2012 Waters Corporation 62

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ResultsResults

Column : HSS C18 3x50mm 1.7µm100% CO2Pressure gradient : from 100 to 180

2

Pressure gradient : from 100 to 180

1

35

4 6

1 naphtalene2 acenaphtene3 fluorene

©2012 Waters Corporation 63

4 2methylnaphtalene5 phenantrene6 fluoranthene

Page 64: ©2012 Waters Corporation 1 · Ir(fppy)3 N F F N AU 0.06 N F F Ir F F N F Ir(fppy)3 Isomer Ir(fppy)3 -F Ir(fppy)3-2F 1.935 6 0.03 N F F F Ir N F N F Ir N F F N F F I (f )3 F Ir N

Customer XCustomer XSample Sample –– UPC²UPC²

0.66

8

5.36

7

0.230

0.240

0.250

pp

M th 60 k

0 160

0.170

0.180

0.190

0.200

0.210

0.220 More than 60 peaks integrated (220 nm)— 30 with % area > 0.1%

— 11 with % area > 0 5%

21

AU

0 100

0.110

0.120

0.130

0.140

0.150

0.160 11 with % area > 0.5%

0.60

5

1.96

1 3.74

1

5.12

0 040

0.050

0.060

0.070

0.080

0.090

0.100

0.43

00.

450

0.46

10.

488

0.85

30.

869

1.83

7

2.04

92.

220

2.27

7

2.49

82.

645

2.71

32.

818 2.

867

2.94

12.

973

3.00

53.

070

3.11

83.

180

3.30

73.

319

3.34

23.

371

3.43

43.

472

3.55

73.

589

3.80

43.

831

3.88

63.

929 3.96

34.

118

4.14

4 4.24

84.

296

4.34

94.

381 4.

476

4.54

14.

586

4.68

74.

704

4.74

74.

766

4.81

14.

836

4.95

64.

997

5.03

95.

164

5.30

15.

479

5.57

1

5.89

85.

969

6.21

16.

270

6.51

9

6.93

1

7.50

5

-0.010

0.000

0.010

0.020

0.030

0.040

©2012 Waters Corporation 64

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 7.50 8.00 8.50 9.00 9.50 10.00

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Zoom on Zoom on baselinebaseline (220nm)(220nm)

0.009

( )( )

Noise is at very low level

0.005

0.006

0.007

0.008

U 0.000

0.001

0.002

0.003

0.004

AU

-0.004

-0.003

-0.002

-0.001

0.000

-0.008

-0.007

-0.006

-0.005

©2012 Waters Corporation 65

-0.009

Minutes0.50 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

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UPLC IUPLC I--Class SeparationClass SeparationBEH Shield RP18; 2.1mm X 100 mmBEH Shield RP18; 2.1mm X 100 mm;;

0.80

1.00 Analytical conditions:— Eluent A: Formic acid 0.1%

AU

0.20

0.40

0.60 — Eluent B: Acetonitrile

— 25% to 100% B in 15 min at 0.5 ml/mn

— Column temperature: 35C0.00

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00

0.10

Column temperature: 35C

More than 60 peaks integrated (285 nm)— 31 with % area > 0.1%

AU

0.04

0.06

0.08 — 12 with % area > 0.5%

0.00

0.02

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00

©2012 Waters Corporation 66

Minutes

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MethodMethod Station vs. UPC²Station vs. UPC²

6.0e+1

Method Station Viridis 2EP 5µm

AU

2.0e+1

4.0e+1

0.66

8

7

0 . 2 4 0

0 . 2 5 0

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

0

5.36

7

0 . 1 4 0

0 . 1 5 0

0 . 1 6 0

0 . 1 7 0

0 . 1 8 0

0 . 1 9 0

0 . 2 0 0

0 . 2 1 0

0 . 2 2 0

0 . 2 3 0 UPC²Acquity BEH 2EP 1.7µm

0.60

5

1.96

1 3.74

1

5.12

1

AU

0 . 0 4 0

0 . 0 5 0

0 . 0 6 0

0 . 0 7 0

0 . 0 8 0

0 . 0 9 0

0 . 1 0 0

0 . 1 1 0

0 . 1 2 0

0 . 1 3 0

©2012 Waters Corporation 67

0.43

00.

450

0.46

10.

488

0.85

30.

869

1.83

7

2.04

9

2.22

02.

277

2.49

8

2.64

52.

713

2.81

8 2.86

72.

941

2.97

33.

005

3.07

03.

118

3.18

0

3.30

73.

319

3.34

23.

371

3.43

43.

472

3.55

73.

589

3.80

43.

831

3.88

63.

929 3.96

3

4.11

84.

144 4.24

84.

296

4.34

94.

381 4.

476

4.54

14.

586

4.68

74.

704

4.74

74.

766

4.81

14.

836

4.95

64.

997

5.03

9

5.16

4

5.30

1

5.47

95.

571

5.89

85.

969

6.21

16.

270

6.51

9

6.93

1

7.50

5

- 0 . 0 1 0

0 . 0 0 0

0 . 0 1 0

0 . 0 2 0

0 . 0 3 0

M in u t e s0 . 0 0 0 . 5 0 1 . 0 0 1 . 5 0 2 . 0 0 2 . 5 0 3 . 0 0 3 . 5 0 4 . 0 0 4 . 5 0 5 . 0 0 5 . 5 0 6 . 0 0 6 . 5 0 7 . 0 0 7 . 5 0 8 . 0 0 8 . 5 0 9 . 0 0 9 . 5 0 1 0 .0 0