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
0.055
0.060
0.065
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
0.010
0.020
0.030
0.040
0.050
0.060
0.070
0.080
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
0.035
0.040
0.045
0.050
0.055
0.060
0.065
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
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
0.010
0.020
0.030
0.040
0.050
0.060
0.070
0.080
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
AU
0.000
0.010
0.020
0.030
0.040
0.050
0.060
0.070
0.080
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
0.010
0.020
0.030
0.040
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)