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Transcript of Co s mos 2014_ruchi
CoSMoS 2014Method Development Olympics
Pfizer, Groton
Analytical R & D Team
1
Team Members
Doug Farrand
Mengtan Zhang
Ron Morris
Ruchi Mehta
Tasneem Patwa
2
Challenge
Goal: To identify and quantitate the main
ingredient in a low dose nasal formulation.
Bonus: To identify and quantitate other
constituents in the formulation.
Materials Provided: 7 x 125 µL sample units and
2 x 125 µL placebo units of the formulation.3
Part 1: Identification
4
Identification by MS
• Components identified by a series of high and low resolution mass spectrometry experiments
• MS data was acquired using a Bruker Solarix XR mass spectrometer in positive electrospray mode
• The structure elucidation predictions made by High (XRMS) and Low (SQD) resolution MS are consistent and exactly overlap with the structure of Hydrocortisone 5
UV and MS Spectrum
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Full-Scan Mass Spectrum
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Meas. m/z Ion Formula m/z err [ppm] rdb
363.217010 C21H31O5 363.216601 -1.1 6.5
Hydrocortisone
8
(11β)-11,17,21-trihydroxypregn-4-ene-3,20-dione
MW = 362.20; Molecular Formula: C21H30O5
NMR
• Instrument: Bruker-Biospin AVANCE III NMR spectrometer operating at 600 MHz
• Solvent: DMSO-d6 with TMS as NMR reference and Maleic Acid as internal standard
• Compared HNMR spectrum of Hydrocortisone Standard versus the formulation
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NMR Spectrum
10
Formulation
HC Standard
Identification of Other Components
• Identified other components by LC-MS• LCMS helped establish the structure of four
other constituents• Main component Hydrocortisone – likelihood
that other components were related to it.• The four other constituents were identified by
MS and confirmed by literature search
11
Other Components1) Cortisone
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Combined - SQ 1: MS Scan 1: 150.00-1000.00 ES+, Centroid, CV=30
169.1
304.3
361.3
Inte
nsity
0.0
5000.0
10000.0
15000.0
20000.0
25000.0
30000.0
35000.0
m/z
200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00 1000.00
Corti
sone
- 2.
651
AU
-0.10
-0.08
-0.06
-0.04
-0.02
0.00
0.02
0.04
0.06
0.08
0.10
Minutes
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 3.20 3.40 3.60 3.80 4.00
Other Components1) Cortisone
• A m/z 361.3 was obtained for the peak at RT 2.639 min. • The UV spectrum for this peak is similar to that of
hydrocortisone, which suggested it, was a closely related steroidal compound.
• Cortisone has a mass of 360.4 (which is consistent with m/z 361.3) and is a common degradation product of Hydrocortisone.
• This constituent was then confirmed to be Cortisone by comparing its RT to the RT of a Cortisone standard obtained from the market.
13
Other Components2) Prednisone
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Combined - SQ 1: MS Scan 1: 150.00-1000.00 ES+, Centroid, CV=30
169.2
327.3
359.3
Inte
nsity
0.0
5000.0
10000.0
15000.0
20000.0
25000.0
30000.0
35000.0
40000.0
m/z
200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00 1000.00
Pre
dnis
one
- 2.
439
AU
-0.020
-0.015
-0.010
-0.005
0.000
0.005
0.010
0.015
0.020
Minutes
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 3.20 3.40 3.60 3.80 4.00
Other Components2) Prednisone
• A m/z 359.2 was obtained for the peak at RT 2.429 min.
• The UV spectrum for this peak is similar to that of hydrocortisone and cortisone, which suggested it, was a closely related steroidal compound.
• Prednisone has a mass of 358.4 (which is consistent with m/z 359.2).
• This constituent was then confirmed to be Prednisone by comparing its RT to the RT of a Prednisone standard.
15
Other Components3) Prednisolone/21-al impurity
• A m/z 361.2 was obtained for the peak at RT 3.057 min. • The UV spectrum for this peak is similar to that of
hydrocortisone, which suggested it, was a closely related steroidal compound.
• Two possibilities:
1) The 21-aldehyde derivative of hydrocortisone - mass of 360.2 (which is consistent with m/z 361.2) and is a common degradation product of Hydrocortisone. Seen in Hydrocortisone Standard as well!
2) Prednisolone – also has a mass of 360.4 (which is also consistent with m/z 361.2)
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3) Prednisolone/21-al impurity
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Combined - SQ 1: MS Scan 1: 150.00-1000.00 ES+, Centroid, CV=30361.2
Inte
nsity
0.0
10000.0
20000.0
30000.0
40000.0
50000.0
60000.0
m/z
200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00 1000.00
3.118
3.294
AU
-0.005
-0.004
-0.003
-0.002
-0.001
0.000
0.001
0.002
0.003
0.004
Minutes
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 3.20 3.40 3.60 3.80 4.00
21-al impurity
Prednisolone
O
OH
H
H H
OH O
O
Structure Comparison
18
Hydrocortisone Cortisone Prednisone
Prednisolone
O
OH
H
H H
OH O
O
21-al impurity
UV Spectrum of Components
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2.455 Peak 1
242.7
338.7 367.9 392.8
AU
0.000
0.001
0.002
2.662 Peak 2198.8 242.7
296.7 311.5 348.1 390.3AU
-0.001
0.000
0.001
0.002
3.098 Peak 3198.8 247.6
313.9 350.5 367.9 390.3
AU
0.000
0.002
0.004
3.280 Peak 4247.6
AU
0.00
0.02
0.04
0.06
nm
200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
Prednisone
Cortisone
Prednisolone
Hydrocortisone
Other Components4) Polyethylene Glycol
20
4) Polyethylene Glycol
21
4) Polyethylene Glycol
• The characteristic umbrella pattern is typical of polyethylene glycol molecules.
• The MS pattern shows fragments that are 44 mass units apart, which also confirms the presence of PEG.
• The masses are around the range of 400 Da, which confirmed that this component was PEG 400.
22
Part 2: Quantitation
23
Dissolving Solvent
• In-silico prediction tool “Cosmotherm”• Highly hydrophobic neutral compound with some polar
groups• Good solubility in organic solvents such as Ethanol, Methanol,
THF, and 50/50 mixtures of aqueous and organic solvents• Explored different ratios of Ethanol/Water (30/70 and 50/50)
and Methanol/Water (50/50) and also Acetonitrile/Water (50/50)
• Relatively low solubility in Acetonitrile• Chosen dissolving solvent: 50/50 mixture of Ethanol/Water
24
Chromatographic Method
• Approach: develop a fast, efficient, accurate, precise, robust, and simple method.
25
Achiral UPLC Screen
26
System Used: Waters Acquity with PDA detector and solvent switcher Time %Buffer %Acetonitrile Flow Rate: 0.5 ml/min 0.00 95 5 Column Temp: 45° C 8.2 0 100 Inj Vol: Varies from 1-5 μl 8.7 0 100 Detection: UV-210 nm (200-400 nm collected) 8.8 95 5 (10pts/sec with 4.8 nm bandwidth) 10.30 95 5 Method Buffer Column 1 50mM Sodium Perchlorate Waters BEH C8 2.1 x 100mm 1.7 um With 0.1% Phosphoric Acid (pH ~2.1) 2 10mM Ammonium Bicarbonate Waters BEH C8 2.1 x 100mm 1.7 um (pH ~8.0) 3 0.1% Methanesulfonic Acid Waters BEH RP C18 2.1 x 100 mm 1.7 um (pH ~2) 4 0.1% Methanesulfonic Acid Waters HSS T3 2.1 x 100 1.8 um (pH ~2)
Not Good Enough!
• None of the screening methods was able to efficiently separate the components.
• All the screening methods utilized Acetonitrile as the organic portion of the mobile phase.
• Since all of the components are steroidal in nature, there is a high degree of hyrophobicity associated with them.
• Replaced Acetonitrile with a stronger organic solvent. • A 2:1 mixture of THF and Methanol was used as the organic
portion of the mobile phase. Different proportions of aqueous to organic were evaluated to obtain the best chromatographic profile
27
Optimized Chromatographic Conditions
CHROMATOGRAPHIC CONDITIONS
Chromatographic system: Waters Acquity UPLC
Column: RP shield C18, 1.8 µm, 2.1 x 100 mm
Column Temperature: 45 °C
Injection Volume: 2 uL
Flow Rate: 0.4 mL/min
Detection: UV @ 245 nm
Mobile Phase:Isocratic
A (77%): 0.05% MSA in Water
B (23%): THF:Methanol (2:1)
Dissolving Solvent: Ethanol : Water (50:50)
Run-Time 4.0 min
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Validation ParametersParameter Method Result
Linearity 0.05mg/ml- 0.13mg/ml) R² = 1.0
LOQ 0.3% of nominal Recovery = 91%
System Precision 5 injections at nominal (0.08mg/ml)
RSD = 0.8%
Method Precision 3 Assays of Sample RSD = 3.9%
Solution Stability (Standard and Sample)
24 hour time point Std recovery= 100.2%Spl recovery= 98.9% of initial time point
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Chromatograms
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Hydro
cort
ison -
3.2
20
AU
-0.15
-0.10
-0.05
0.00
0.05
0.10
0.15
Minutes
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 3.20 3.40 3.60 3.80 4.00
Cort
isone -
2.6
51
AU
-0.10
-0.08
-0.06
-0.04
-0.02
0.00
0.02
0.04
0.06
0.08
0.10
Minutes
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 3.20 3.40 3.60 3.80 4.00
Pre
dnis
one -
2.4
39
AU
-0.020
-0.015
-0.010
-0.005
0.000
0.005
0.010
0.015
0.020
Minutes
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 3.20 3.40 3.60 3.80 4.00
Pre
dnis
one -
2.4
25
Cort
isone -
2.6
36
Pre
dnis
olo
ne -
3.0
53
Hydro
cort
ison -
3.2
21
AU
-0.050
-0.040
-0.030
-0.020
-0.010
0.000
0.010
0.020
0.030
0.040
0.050
Minutes
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 3.20 3.40 3.60 3.80 4.00
Hydrocortisone
Cortiosone
Prednisone
Sample
31
Hydrocortisone Linearity
Quantitation Protocol• After successfully validating, and comparing sample responses
to the curve, nominal concentration for Hydrocortisone was set at 0.08mg/mL
• Cortisone Standard: Prepared a standard of Cortisone at 0.002678 mg/ml
• Prednisone Standard: Prepared a standard of Prednisone at 0.002312 mg/ml
• Prednisolone was quantitated against hydrocortisone standard.
• Quantitation was performed by preparing 3 individual samples• Samples were prepared by diluting about 100mg of
formulation in 1ml volumetric flasks to obtain area responses comparable to that of the standard at nominal concentration.
32
Assay Results
33
Component Sample 1 Sample 2 Sample 3Calculated Assay (%w/w)
Concentration (mg/ml)
Assay % of Theoretical
Hydrocortisone 697822 661686 609220
0.1187% 0.1103% 0.1176% 0.1155% 1.2 mg/ml 93%
Cortisone 28646 27191 11689
0.0047% 0.0043% 0.0043% 0.0044% 0.044 mg/ml 94%
Prednisone 16703 16406 7253
0.0027% 0.0028% 0.0029% 0.0029% 0.030 mg/ml 94%
Prednisolone 41265 39478 17750
0.0075% 0.0075% 0.0074% 0.0075% 0.076 mg/ml 88%
Comparison with USP Method
Comparison CoSMoS Method USP Method
Instrument time 4.0 min per injection 15.0 min per injection
Organic solvent consumption per sample
0.5mL per sample 1mL per sample
Organic solvent consumption per injection
0.37mL 7.5mL
Cost of column $400 $400
Total Time Say “T” Approx “4T”
Total Cost Say “C” Approx “ 20C”
34
ConclusionComponents Concentration Assay % of Theoretical
Hydrocortisone 1.2 mg/ml 93%
Cortisone 0.044 mg/ml 94%
Prednisone 0.030 mg/ml 94%
Prednisolone 0.076 mg/ml 88%
PEG 400 N/A N/A
Water 19.5% w/w 91%
35
Conclusion• The main component of the low dose formulation was identified
to be Hydrocortisone (by HRMS and NMR)• Six out of the Nine components were identified and quantitated• Simplified sample preparation
A simple dissolving solvent Water/Ethanol (50/50) Short sample preparation time
• Rapid UHPLC method for Quantitation Reduced run-time on the instrument within 4.0 min Highly cost effective Accurate and Precise Green
36
AcknowledgmentsAngel Diaz
Mike Coutant
Zhaohui Lei
David Foley
37
Thank You!!!
38
Questions???
39