Achiral SFC: Development of an Orthogonal SFC … Wang Merck Research Laboratories Achiral SFC: Development of an Orthogonal SFC Method for Mometasone Furoate Impurity Analysis NJCG

Zhenyu WangMerck Research Laboratories

Achiral SFC:

Development of an Orthogonal SFC Method for

Mometasone Furoate Impurity Analysis

NJCG 2013

Outline

• SFC chronicleThe journey of a half century

• Re-birth of SFCChiral SFC in pharmaceutical industry

• New opportunity of SFC eraAchiral SFC for complex analysis

• Summary

The chronicle of SFC1962-1981: Packed Column SFC

1962:Ernst Klesper

“High pressure GC”

1981:M. Novotny, and M.L. Lee. et.al.

The chronicle of SFC1981-1995: Capillary SFC

1980s: the enthusiasm

“…SFC should exhibit the combined advantages of GC and HPLC.”

“SFC…allows the separation of materials which are thermally labile and of much higher molecular weight than is possible using GC.”

“High diffusivities, low viscosities SF CO2 provide higher flow rates, shorter analysis, and higher efficiency separations.”

1990s: the frustrations

“The extent to which SFC will be useful for relatively polar materials is still an open question and a matter for much future research.”

A decade debate: “Open tubular SFC” vs. “Packed column SFC”

The winner: Packed column SFC: user friendly, quantitative

injection, and apply to higher polarity compounds

Application:Chiral separation and purificationPolar compounds even peptides

Hardware:Dedicated SFC stationary phasesNew generation SFC instrumentation

Conception: Green technologyCost effective

The chronicle of SFCLate 1990s-present: Re-birth of parked column SFC

Ref.: Jeff Elleraas, Pfizer

Key driver of SFC’s RenaissanceChiral separation and purification in Pharm

Chiral Prep SFC : Stacked injections 16.5 g in 7 hours

400350300250200150100500

2,100

2,000

1,900

1,800

1,700

1,600

1,500

1,400

1,300

1,200

1,100

1,000

900

800

700

600

500

400

300

200

100

0

-100

Chiral SFC for non-chiral compound separation

O

O

OMeMeO

OMe

MeO

OMe

OMe

O

O

OH

OMeMeO

MeOOMe

OMe

• Nobiletin (NOB): anti-inflammatory agent

• NOB is metabolized by hepatic p450 enzymes yielding 3’-demethyl-

NOB and 4’-demethyl-NOB

• No separation of the two metabolites on RPLC

Nobiletin (NOB)

O

O

OH

OMeMeO

OMe

MeOOMe 4’-demethyl-NOB

3’-demethyl-NOB P450

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00Time0

100

%

Z5800503 ELSDAn1

7.70e53.87

No Separation on Reversed Phase LC

3’-DNOB

4’-DNOB

Mass Spectrum:

RPLC separation of 3’-DNOB and 4’-DNOB

SFC: 20% MeOH as modifier, 2.0 mL/min, CO2 100 bar, 30ºC

Column: Chiralpak AD

20181614121086420

50403020100

AD0420055.DATA [1] RT [min]mAU SFC

Rs: 22.5

Rs: 1.7

LC

3’-DNOB and 4’-DNOB: Chiral LC vs. Chiral SFC

HPLC: 40/60 hexane/ethanol, 1.0 mL/min

Column: Chiralpak AD

Chiral SFC for non-chiral compound separation

252423222120191817161514131211109876543210

3432302826242220181614121086420

-2

AD04200516.DATA [1] RT [min]mAU

4’-Demethyl-NOB(major metabolite)

28.9 µg/mL

3’-Demethyl-NOB(minor metabolite)

ID and quantitation of NOB metabolites in mice urine

SFC: AD column, 20% MeOH as modifier, 2.0 mL/min, CO2 100 bar, 30ºC

NOB(parent)

Z. Wang, S. Li, Biomed. Chromatogr., 20 (2006) 1206-1215

The new opportunity of SFC era: Achiral separation of complex analytes

SFC achiral method development• General practice for method development

• Mometasone furoate case study

SFC achiral method validation• Improve method sensitivity

SFC sample prep for complex matrix (e.g. bio-fluids,

formulated drugs)

Mometasone furoate franchise

A highly potent glucocorticoid anti-inflammatory agent The active ingredient of several drug products

Mometasone furoate and its major impurities

O

Cl

HOO

O

O

O

Cl

Mometasone furoate

Current RPLC method for MF impurity analysis

8.16

5

11.3

90

18.6

88

22.5

00

24.8

25

27.4

87

32.8

47 36.9

29

40.3

22

AU

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

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0.060

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0.070

0.075

0.080

Minutes2.00 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 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00

Sample: MF with spiked impuritiesColumn: Ultrasphere ODS (250 × 4.6 mm, 5 µm)Mobile phase A: Water/Mobile phase B: Acetonitrile Gradient: 42% B to 52% B in 60 min Flow rate: 1.5 mL/min

*Method slightly modified from USP MF monograph

What if I use SFC

42 min !

SFC & HPLC instrument and software

All SFC experiments were performed on:TharSFC Method Station Analytical System

– Solvent selector– Column selector– Waters 2998 PDA detector

Instrument control and data collection: Empower 2

All HPLC experiments were performed on:Alliance 2690 HPLC System equipped with 2996 PDA detectorInstrument control and data collection: Empower 2

Achiral SFC method development

Preliminary Screening:

Stationary Phase & Modifier

2° Screening:

P, T, Additive

Fine tuning

SFC achiral column screening

• Most time-consuming step

• Retention mechanism is not fully understoodNormal phase,

Reversed phase,

Mixed mode

• Simplify column screening

SFC column selection: QSRR: Quantitative Structure Retention Relationship

West, C.; Lesellier, E., J. Chromatogr. A, 2008, 1203, 105.

Primary screening: Column and Modifier

MeOHEtOH2-PrOH

1.100

1.075

1.050

1.025

1.000

SiCyano2-EP

1.100

1.075

1.050

1.025

1.000

Column

Modifier

2-EPCyanoSi

Column

2-PrOHEtOHMeOH

Modifier

Interaction Plot for SelectivityData Means

Separation on Silica column with Methanol as modifier

4.84

15.

095

5.91

2

7.73

0 8.34

7 8.64

99.

078

9.54

6

11.4

89

AU

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

8

57 1

6

2

3 4

MF

Achiral SFC method development – Step Two

Preliminary Screening:

Stationary Phase & Modifier

2° Screening:

P, T, Additive

Fine tuning

Impact of temperature and pressure

4.84

15.

095

5.91

2

7.73

0 8.34

7 8.64

9

9.07

8

9.54

6

11.4

89

AU

0 . 0 0 0

0 . 0 1 0

0 . 0 2 0

0 . 0 3 0

0 . 0 4 0

0 . 0 5 0

0 . 0 6 0

0 . 0 7 0

0 . 0 8 0

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

5.47

4

5.79

2

6.66

9

8.53

8

9.24

4 9.44

2

9.88

0

10.2

27

12.3

43

AU

0 . 0 0 0

0 . 0 1 0

0 . 0 2 0

0 . 0 3 0

0 . 0 4 0

0 . 0 5 0

0 . 0 6 0

0 . 0 7 0

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

Silica: 250 × 4.6 mm, 5 µm, Kromasil Mobile phase: 4 mL/min, CO2

Gradient: 5% MeOH to 20% MeOH in 15 min

35 ºC, 100 bar

30 ºC, 100 bar

Impact of temperature and pressure (Cont’d)

Silica: 250 × 4.6 mm, 5 µm, Kromasil Mobile phase: 4 mL/min, CO2

Gradient: 5% MeOH to 20% MeOH in 15 min

6.01

9

6.33

5

7.24

8

9.25

0

10.1

77

10.6

56

13.2

86

AU

- 0 . 0 0 5

0 . 0 0 0

0 . 0 0 5

0 . 0 1 0

0 . 0 1 5

0 . 0 2 0

0 . 0 2 5

0 . 0 3 0

0 . 0 3 5

0 . 0 4 0

0 . 0 4 5

0 . 0 5 0

0 . 0 5 5

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

40 ºC, 100 bar

Impact of temperature and pressure (Cont’d)

Silica: 250 × 4.6 mm, 5 µm, Kromasil Mobile phase: 4 mL/min, CO2

Gradient: 5% MeOH to 20% MeOH in 15 min

4.10

84.

339 4.79

7

6.56

66.

605

6.77

3

7.47

9

7.87

5

8.48

3

10.2

61

AU

0 . 0 0 0

0 . 0 1 0

0 . 0 2 0

0 . 0 3 0

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

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

30 ºC, 150 bar

4.84

15.

095

5.91

2

7.73

0 8.34

7 8.64

9

9.07

8

9.54

6

11.4

89

AU

0 . 0 0 0

0 . 0 1 0

0 . 0 2 0

0 . 0 3 0

0 . 0 4 0

0 . 0 5 0

0 . 0 6 0

0 . 0 7 0

0 . 0 8 0

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

30 ºC, 100 bar

4.84

15.

095

5.91

2

7.73

0

8.34

7 8.64

9

9.07

8

9.54

6

11.4

89

AU

0.000

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Minutes1.00 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 15.00

8

5 71

6

2

3 4

MFSFC

RPLC

8.16

5

11.3

90

18.6

88

22.5

00

24.8

25

27.4

87

32.8

47 36.9

29

40.3

22

AU

0.000

0.005

0.010

0.015

0.020

0.025

0.030

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0.050

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0.060

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0.070

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0.080

Minutes2.00 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 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00

Good news for pharmaceutical chromatographers

One of the most challenges for us:

• To develop stability indicating method to monitor DS & DP

Most of stability indicating methods are RPLC based

How to confirm method’s specificity (detection or separation)

SFC can be the 2nd method with true orthogonal selectivity

I get new tool

Stability indicating method: RPLC or SFC?

Why don’t we use SFC for the primary

stability indicating method?

ButIs SFC sensitive enough?

Which one?

Sensitivity: key to drug impurity analysis

Drug safety and quality control

Reporting threshold: 0.05% impurity in Drug Substance (≤ 2 g/day), ICH Q3A

Reporting threshold: 0.1% degradation product in Drug Product (≤ 1 g/day), ICH Q3B

Why SFC-UV is less sensitive (vs. HPLC-UV)

Three main sources of noise:

Electronic: noise from detector system

Mechanical: BPR, pump

Thermal: endothermic process during depressurization

Anton, K.et.al. Analusis, 1999, 27, 691Helmy, R. et.al. Chirality, 2007, 19, 787Wang, Z, et.al, Am. Pharm. Rev, 2009, 5, 59

How to get better sensitivity on SFC-UV

Software filtering: reduce non-wavelength dependent noise 1,2

Hardware modification: reduce mechanical and thermal noise 3

Next generation SFC

1 Chen, R., LC-GC Application Notebook, 2009, Sep2. Wang, Z, et.al, J. Chromatogr. A, 2011, 1218, 23113. Helmy, R. et.al. Chirality, 2007, 19, 787

PDA detector settings Without wavelength compensation

With wavelength

compensation

Improvement in sensitivity

Sampling rate

Bandwidth Filter constant

Peak width S/Na Peak width S/Nb (S/Nb)/(S/Na)

i 5 2.4 Slow 0.072 67 0.072 151 2.2ii* 5 2.4 Normal 0.066 44 0.066 139 3.2iii* 5 3.6 Slow 0.073 57 0.072 163 2.9iv 5 3.6 Normal 0.066 62 0.066 122 2v 5 4.8 Slow 0.072 62 0.072 142 2.3vi 5 4.8 Normal 0.066 54 0.067 115 2.1

vii* 2 2.4 Slow 0.108 62 0.108 251 4viii 2 2.4 Normal 0.077 71 0.077 170 2.4ix 2 3.6 Slow 0.108 52 0.108 190 3.7x 2 3.6 Normal 0.077 47 0.077 159 3.4xi 2 4.8 Slow 0.109 63 0.108 175 2.8xii 2 4.8 Normal 0.077 43 0.077 162 3.8

Reference Wavelength Compensation to increase S/N

Detection wavelength: 245 nm

Compensation wavelength: 400-450 nm

AU

0.000

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0.020

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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 15.00

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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

ii

iii

AU

0.000

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0.020

0.030

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0.050

0.060

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

vii

Ref. Wavelength Compensation to increase S/N (Cont’d)

Sampling rate: 5Bandwidth: 2.4Filter cons.: NormalPeak width: 0.066S/N: 139

Sampling rate: 5Bandwidth: 3.6Filter cons.: SlowPeak width: 0.072S/N: 163

Sampling rate: 2Bandwidth: 2.4Filter cons.: SlowPeak width: 0.108S/N: 251

S/N x3 increase

Comparison of RPLC and SFC method validation results

RP-HPLC method SFC method

Sample concentration 0.2 mg/mL 2.0 mg/mL

Linearity 0.9999 0.9999

Accuracy Assay levela 99.1% - 100.7% 99.8% -101.6%

Impurity levelb 96.6% - 115.4%c 88.3% - 104.7%c

Precision Assay levela 0.4% 0.7%

Impurity levelb 1.9% - 5.0% 1.4% - 5.4%

Limit of Quantitation 0.05% (or 0.1 µg/mL) 0.05% (or 1.0 µg/mL)

a. Six preparations (n = 6)b. Six preparations of spiked individual impuritiesc. The average recovery of each impurity was reported.

Z. Wang, J. Chromatogr. A, 2011, 1218, 2311-2319

Sensitivity is not a major challenge for SFC anymore…

Can new instrumentation broaden SFC to complex sample analysis? Such as formulated drugs.

Using SFC in complex formulation analysis

The Big Fact:SFC is rarely used for drug product analysis

Chromatograph used for drug product analysis: RPLC, nearly 100%

The Challenge:

Drug product sample prep: working for RPLC ≠ working for SFC

SFC prefers neat organic as sample solvent

Direct injection of aqueous containing sample on SFC may result in:

- Freezing of aqueous solutions

- Precipitation of samples

- Deteriorated separations

SFC Sample Preparation Strategies for Drug Products

For solid dosage forms:

- Dissolve sample in organic solvent

- Filter to remove un-dissolved residue

- Assay by SFC

Extraction robustness need to be assessed

For solutions and suspensions:

- Direct injection might work (not recommended as 1st choice)

- Use Solid Phase Extraction or Liquid-Liquid Extraction

SPE-SFC: for an aqueous formulation

0

20

40

60

80

100

120

(%)

Strata

X

C18 E C8

Pheny

l

SDB-L

SPE Cartridge Screen Study

Methanol DMSO Acetonitrile

SFC

Rec

over

y (%

)

Z. Wang, Am. Pharm. Rev, 2013, 16(3), 28-25

Summary

SFC dominated chiral separation and chiral purification

The advantages of achiral SFC have not been fully recognized:

Orthogonal selectivity

Economical

Faster

SFC friendly sample prep is important for complex analysis (e.g. for

formulated drugs)