Mdo2014 takeda

CoSMoS Method Development Olympics 2014
11August2014
Amy McCusker
Research Investigator
Analytical Development Laboratories, CMC Center
Takeda Pharmaceuticals International Co.

Outline
• Introduction
– Myself
– Strategy
• API Identification
– Parent peak ID by LCMS
– Confirmation by HRMS
– Confirmation by NMR
• Quantitation
– Ions (IC)
– Volatiles (GC/FID)
– UHPLC Assay
• Polymers
• API and related substances
• Post-challenge notes
CosMos Method Development Olympics 2014 11Aug20141

Introduction
• Takeda Pharmaceuticals International Co. (Cambridge, MA)
The opinions I present here are my own and do not represent those of my employer, Takeda Pharmaceuticals International Co.
• Why MDO?
• Cross-functional collaboration
• Interesting way to participate
with the scientific community
• I wanted to come to CoSMoS
• Fun

Strategy
Plan:
1. Identification
a) Assess
b) Obtain standards
c) Confirm
2. Assay development
a) Choose appropriate
instrumentation / detector
b) pH screen
c) Stationary phase screen
3. Identify and quantitate other
constituents
a) IC screen for salts
b) GC screen for volatiles
Goals:
1. Primary: solve the challenge
a) Use existing methods
b) Instruments available:
• UPLC/TQD, UPLC/SQD,
UPLC/HRMS , GC/MS, GC/qTOF,
FTIR/Raman/nIR, NMR
2. Secondary
a) Other constituents
b) Method assessment
Challenges:
1. Sample Type
2. Sample Information
3. Time!

Outline
• Introduction
– Myself
– Strategy
• Quantitation
– Ions (IC)
– UHPLC Assay
• Polymers

API Identification
1. Appearance:
– Packaging:
• small glass vials plugged with rubber septa.
– Drug product:
• Highly viscous
• Clear
• Colorless
2. Smell / Color:
– Not fluticasone!
3. UHPLC/MS
– Waters Acquity / Xevo TQD and PDA
– Default UPLC/MS method conditions (0.1% FA in
water / ACN, Acquity BEH C18 Column)
– Sample and placebo diluted with water, 10x
volumetrically
– Detection:
• Full ESI scan in (+) and (-) modes
• Full PDA scan

API Identification: MS Analysis
First chromatogram (UV at 254 nm)
Placebo
Sample
Masses for API peak detected on TQD:
• ESI (-) mode:
– Major: 407.1 m/z
– 769.7 m/z
• ESI (+) mode:
– Major: 363.3 m/z
– 725.6 m/z

Reference:
http://fiehnlab.ucdavis.ed
u/staff/kind/Metabolomic
s/MS-Adduct-Calculator
Table 1. Monoisotopic exact masses of molecular ion adducts
often observed in ESI mass spectra Your M here:
362.30000
Ion name Ion mass Result:
1. Positive ion mode
M+3H M/3 + 1.007276 121.773943
M+2H+Na M/3 + 8.334590 129.101257
M+H+2Na M/3 + 15.7661904 136.532857
M+3Na M/3 + 22.989218 143.755885
M+2H M/2 + 1.007276 182.157276
M+H+NH4 M/2 + 9.520550 190.670550
M+H+Na M/2 + 11.998247 193.148247
M+H+K M/2 + 19.985217 201.135217
M+ACN+2H M/2 + 21.520550 202.670550
M+2Na M/2 + 22.989218 204.139218
M+2ACN+2H M/2 + 42.033823 223.183823
M+3ACN+2H M/2 + 62.547097 243.697097
M+H M + 1.007276 363.307276
M+NH4 M + 18.033823 380.333823
M+Na M + 22.989218 385.289218
M+CH3OH+H M + 33.033489 395.333489
M+K M + 38.963158 401.263158
M+ACN+H M + 42.033823 404.333823
M+2Na-H M + 44.971160 407.271160
M+IsoProp+H M + 61.06534 423.365340
M+ACN+Na M + 64.015765 426.315765
M+2K+H M + 76.919040 439.219040
M+DMSO+H M + 79.02122 441.321220
M+2ACN+H M + 83.060370 445.360370
M+IsoProp+Na+H M + 84.05511 446.355110
2M+H 2M + 1.007276 725.607276
2M+NH4 2M + 18.033823 742.633823
2M+Na 2M + 22.989218 747.589218
2M+3H2O+2H 2M + 28.02312 752.623120
2M+K 2M + 38.963158 763.563158
2M+ACN+H 2M + 42.033823 766.633823
2M+ACN+Na 2M + 64.015765 788.615765
Your M here:
2. Negative ion mode 362.30000
Ion name Ion mass Result:
M-3H M/3 - 1.007276 119.759391
M-2H M/2 - 1.007276 180.142724
M-H2O-H M- 19.01839 343.281610
M-H M - 1.007276 361.292724
M+Na-2H M + 20.974666 383.274666
M+Cl M + 34.969402 397.269402
M+K-2H M + 36.948606 399.248606
M+FA-H M + 44.998201 407.298201
M+Hac-H M + 59.013851 421.313851
M+Br M + 78.918885 441.218885
M+TFA-H M + 112.985586 475.285586
2M-H 2M - 1.007276 723.592724
2M+FA-H 2M + 44.998201 769.598201
2M+Hac-H 2M + 59.013851 783.613851
3M-H 3M - 1.007276 1087.907276
Hypothesis: m/z = 362.3

API Identification: LC/HRMS
• HRMS analysis performed by BPP colleagues
– Provided same diluted sample, mobile phases and method parameters
• Instrument:
– Shimadzu Nexera UHPLC split 5% to Thermo LTQ Orbitrap Velos MS
• ESI (+) 101 – 1500 Da
• Masses detected on HRMS:
– ESI (+) mode:
• M+H : 363.2163 m/z
• 2M+H : 725.4258 m/z
• Proposed chemical formulas calculated by HRMS
• Merck index search result: Hydrocortisone (Formula #2)
M + H Accuracy (ppm)
1. C19H33O2F2S -0.1
2. C21H31O5 -0.7
3. C17H27N4F4 -0.8
4. C16H31O4F4 2.9
5. C22H32OFS 3.0
6. C22H35S2 -3.1

API Confirmation: LC/HRMS/MS
• MS/MS products scan on m/z 363 for unknown sample
• Proposed fragmentation pathway:
AmyUnknow n #1123-1127 RT: 10.15-10.16 AV: 2 NL: 6.61E5
F: FTMS + c ESI d Full ms2 363.20@cid35.00 [85.00-375.00]
100 120 140 160 180 200 220 240 260 280 300 320 340 360
m/z
0
10
20
30
40
50
60
70
80
90
100
327.1968
309.1861
345.2075
267.1750
297.1861
364.2221121.0649
249.1645
279.1752
209.1334187.1124169.1015147.0810
109.0651

API Confirmation: 1H NMR
• Instrument: 500 MHz Bruker Advance III spectrometer
– Predicted spectra:
• Three samples compared:
– 10 μL sample mixed with 50 μL D2O
– 10 μL placebo mixed with 50 μL D2O
– Hydrocortisone standard in 100 μL D2O + 20 μL placebo
• Extra peaks observed in sample spectra:
– 5.10 ppm and above
• Alkyl protons
• No evidence of aromatic protons
• Hydrocortisone standard (Sigma Aldrich Lot SLBJ0126) spectra
matches unknown

API Confirmation: 1H NMR
Placebo spiked with
hydrocortisone standard
Unknown sample
in D2O

Outline
• Introduction
– Myself
– Strategy
• Quantitation
– Ions (IC)
– UHPLC Assay
• Polymers

Anion Identification and Quantitation
• In-house ion chromatography anion method for resolution and
quantitation of six anions
– Dionex ICS5000 with IonPac AS11 Column and KOH eluent generator
(gradient)
– Formate ion identified via RT (RRT = 1.01 to external formate standard)
• 30 mg/mL sample in water calculated against 10 µg/mL external formate
standard
• Reported result: 241 ppm formate (3 preparations)
Unknown
Standard Mix
Blank

Cation Identification and Quantitation
• In-house ion chromatography cation method for resolution and
quantitation of sodium and potassium
– Dionex ICS5000 with IonPacCS11 Column and MSA eluent generator
(isocratic)
– Sodium ion identified via RT (RRT = 1.00 to external sodium standard)
• 30 mg/mL sample in water calculated against 10 µg/mL external sodium
standard
• Reported result: 144 ppm sodium (3 preparations)
Unknown
Standard
Blank

Volatiles Identification
• In-house GC/FID method verified to resolve and quantify 21 residual
solvents:
– Agilent HSGC/FID with DB-624 column (split to MSD for peak confirmation)
– Volatiles identification
• Ethanol identified via RT (RRT = 1.00 to external EtOH standard)
• Library search of TIC peaks identified propylene glycol at 8 minutes
• An unknown peak elutes early
– Agilent column literature lists acetaldehyde as earliest eluter on DB-624
– Identity confirmed via RT by comparison with acetaldehyde / THF solution (RRT = 1.00)
pA
2.00
2.50
3.00
3.50
4.00
4.50
Minutes
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 10.50 11.00 11.50 12.00 12.50 13.00
Ethanol
PropyleneGlycol
Acetaldehyde
Blank
Unknown

Volatiles Quantitation
• Linear range of method is established up to 20,000 ppm (2%) based
on 20 mg/mL sample preparation
– Calibration curve 100 ppm through 5% Ethanol constructed
• Acetaldehyde standard was not obtained
– amount was calculated as % area against ethanol peak
Reported results (2 preparations):

Unknown Identification
Additional peaks visible in spectrum (shortened gradient):
PDA scan
ESI (+) scan
ESI (-) scan
AU
0.00
0.50
1.00
Minutes
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
Intensity
0
5x108
Minutes
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
Intensity
5.0x106
1.0x107
1.5x107
2.0x107
Minutes
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
Whole area extracted and combined

• Extracted ESI (+) spectra: combined peaks over indicated range
– Δ = 44 Da
– Highest abundance = 385.3 Da
Serial No. QCA 581
15:11:06
10-Jun-2014Cosmos_placebo 5x
m/z
100 150 200 250 300 350 400 450 500 550 600 650 700 750
%
0
100
Cosmos_placebo x5_3 799 (5.858) Cm (387:947) 1: MS2 ES+
1.07e7
385.3297
341.2697
297.1640
253.0689
209.0439
165.0056
109.9229
132.9574
429.3407
473.3651
517.3975
561.4229
605.4007
635.3506649.3431
679.3447
693.3751
767.3854

Δ = 44 Da is signature of polyethylene glycol related compounds
Reference: Hui Tong, Duncan Bell, Keiko Tabei, & Marshall M. Siegel (1999). Automated Data
Massaging, Interpretation, and E-Mailing Modules for High Throughput Open Access Mass
Spectrometry,. Journal of American Society for Mass Spectrometry. 10, 1174–1187

• Excipients screened (available from Solid Formulations group):
– Polyethylene Glycol (PEG)
• Average MW = 200, 400, and 3350 screened
– Polysorbate
• 80 and 20 screened (Polysorbate-20 pictured)
– Triton X-100
– Labrasol®
• Proprietary structure composed of PEG esters, glycerides, and free
PEG

• Tween-80
• Placebo
• Triton-X
• Labrasol®
• PEG-400
Serial No. QCA 581
14:11:07
13-Jun-2014Excipient screen
Time
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
%
0
100
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
%
0
100
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
%
0
100
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
%
0
100
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
%
0
100
Tween 5% 1: MS2 ES+
TIC
1.73e8
10.53
10.36
10.00
9.539.228.81
8.32
7.697.40
10.71 11.7011.83
11.93
12.08
12.31
Cosmos_placebo x5_3 1: MS2 ES+
TIC
4.60e8
5.865.51
4.854.30
3.86
3.313.08
0.79
0.46
2.98
2.30
1.521.90
6.28 6.42
6.56
6.58
10.416.89 10.25
9.50
8.157.88
9.268.87 10.64 11.8612.09
Triton-x 2mg 1: MS2 ES+
TIC
1.89e8
11.2310.649.509.427.587.456.93
6.39
5.955.454.99
8.988.15 10.13
12.2111.72 12.45
12.71
12.84
13.06
Labrasol 2mg 1: MS2 ES+
TIC
2.73e8
6.115.82
5.48
5.11
4.68
4.143.89
3.603.211.050.72
2.071.96
6.35
6.807.00
11.60
7.33 11.2710.66
10.289.428.677.74
12.2012.32
12.70
PEG400 3 1: MS2 ES+
TIC
4.29e8
11.636.85
6.226.01
5.73
5.004.58
4.06
3.37
3.24
11.327.00
11.147.16
10.98
7.28
11.82
12.08
12.26
12.36

• PEG-400 ESI (+) spectra extracted 4 min – 9.5 min overlaid with
unknown
Serial No. QCA 581
11:21:38
02-Jul-20146 PEG400 2.025mg
m/z
150 200 250 300 350 400 450 500 550 600 650 700 750 800
%
0
100
6 PEG400 2 917 (6.766) Cm (842:935) 3: MS2 ES+
9.79e6
459.3278
371.1924
327.1373
283.0260
238.9818
194.9689
213.9570 260.9644
415.2563
392.9949
503.2910
547.3471
591.3284
635.3179
679.3762
723.2919
M + H = 283, when n = 6
M + H = 327, when n = 7
M + H = 371, when n = 8
M + H = 415, when n = 9
M + H = 459, when n = 10
M + H = 503, when n = 11
What about these?

• Sigma-Aldrich search for PEG (+Me): Poly(ethylene glycol) methyl ether
– Average MW = 350, 550 purchased
– mPEG 350 ESI (+) spectra extracted 4 min – 9.5 min overlaid with unknown
Serial No. QCA 581
10:33:42
02-Jul-2014mPEG350 2.264mg
m/z
150 200 250 300 350 400 450 500 550 600 650 700 750 800
%
0
00
mPEG350 2 915 (6.689) Cm (835:947) 3: MS2 ES+
9.79e6
385.2866
341.2102
297.1251
253.0425
208.9856
165.0102
186.9116
230.9416 275.0502
429.3125
473.2740
517.2883
561.3499
605.3434
649.3272
710.4086
M + H = 296, when n = 6
M + H = 340, when n = 7
M + H = 384, when n = 8
M + H = 428, when n = 9
M + H = 472, when n = 10
M + H = 516, when n = 11

UHPLC Assay Development and Quantitation
• Scouting run on Acquity looked promising: very little method
optimization was done on the assay
– Propylene glycol is separated
– mPEG and PEG can be quantitated separately via MS
– API is separated from two related substances
– Running out of time!
• Final method parameters:
– Column: Acquity BEH C18, 2.1 mm × 100 mm × 1.7 μm
– Mobile Phase: A = 0.1% Formic Acid, B = 0.1% Formic Acid in ACN
• 0.25 mL/min
• Gradient: 99% A to 50% A, 15 min
– 30 mg/mL sample in water prepared in triplicate
– Detection:
• Full PDA Scan
• ESI (+) Scan 40 – 2000 Da
• ESI (-) Scan 40 – 2000 Da

ESI (+) TIC Scan for final assay
Unknown
Propylene Glycol
mPEG-350
PEG-400
API retention time =
9.1 min

API and Related Substances Quantitation
• 243 nm was selected for quantitation (API maximum λ)
• 30 mg/mL sample quantitated against five replicate injections of 25
μg/mL Hydrocortisone standard
– Sigma Aldrich Lot SLBJ0126V, 98.7% Purity
• Related substances were calculated as % area vs. API
• Reported result (3 preparations):
– 0.12% w/w Hydrocortisone
– 5.7% area vs API for RRT = 0.99
– 4.2% area vs API for RRT = 1.01
Serial No. QCA 581
18:33:31
02-Jul-201430 Unknown 5 57.50mg
Time
-0.00 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
0.0
5.0
1.0e+1
1.5e+1
2.0e+1
2.5e+1
3.0e+1
3.5e+1
4.0e+1
4.5e+1
5.0e+1
5.5e+1
6.0e+1
30 Unknown 5 5: Diode Array
Range: 6.827e+18.99
8.90
0.94
Another impurity is
starting to separate:
needs optimization!
Serial No. QCA 581
18:33:31
02-Jul-201430 Unknown 5 57.50mg
Time
8.78 8.80 8.82 8.84 8.86 8.88 8.90 8.92 8.94 8.96 8.98 9.00 9.02 9.04 9.06 9.08 9.10 9.12 9.14 9.16 9.18 9.20 9.22 9.24
AU
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
Range: 5.801e+1
8.90
9.09

Propylene Glycol Quantitation
• ESI (+) TIC Chromatogram was
used for quantitation for simplicity
(mainly 77 m/z)
• 30 mg/mL sample quantitated
against five replicate injections of
5 mg/mL Propylene Glycol
standard (12.2% RSD)
• Reported Result (3 preparations):
– 8.3% w/w Propylene Glycol
MW = 76.1 g/mol
Unknown
Propylene Glycol

PEG-400 and mPEG-350 Quantitation
• 20 of the most abundant spectral peaks for each material were
analyzed
– Two ions were selected for each based on
• Peak shape
• Consistency
• Selectivity
Serial No. QCA 581Cosmos_placebo 5x
100 150 200 250 300 350 400 450 500 550 600 650 700 7
%
0
100
Cosmos_placebo x5_3 799 (5.858) Cm (387:947)
385.3297
341.2697
297.1640
253.0689
209.0439
165.0056
109.9229
132.9574
429.3407
473.3651
517.3975
561.4229
605.4007
635.3506649.3431
679.3447
693.3751
371.1924
194.9689
213.9570
283.0260
327.1373
415.2563
459.3278
503.3278
547.3471
591.3284
mPEG m/z selected
PEG m/z selected

PEG-400 and mPEG-350 Quantitation
• 30 mg/mL sample quantitated
against five replicate injections of
PEG-400 and mPEG-350 standard
solutions
– 5 mg/mL mPEG selected as
standard
– 2 mg/mL PEG selected as
standard
– 2.9 – 3.9% RSD
• Reported result (3 preparations):
– 28 / 72 mixture of PEG-400 and
mPEG-350
– Total = 28% w/w
Unknown
mPEG-350
PEG-400

Outline
• Introduction
– Myself
– Strategy
• Quantitation
– Ions (IC)
– UHPLC Assay
• Polymers

Reported Result vs Actual Formulation
Component Reported Formulation Actual Formulation
API 0.12% w/w Hydrocortisone 1.316 mg/mL Hydrocortisone
Related Substances
5.7% area vs parent RRT = 0.99
4.2% area vs parent RRT = 1.01
Cortisone: 0.047 mg/mL
Prednisone: 0.032 mg/mL
Prednisolone: 0.086 mg/mL
Excipient 7.8% PEG-400 17% PEG-400
Excipient 20.2% mPEG 49% mPEG
Excipient 8.3% w/w Propylene Glycol 6% Propylene Glycol
Volatile 5.6% w/w Ethanol 7% Ethanol
Volatile
0.29% area vs Ethanol
Acetaldehyde
Not mentioned in formulation
Buffer
140 ppm Sodium ion
240 ppm Formate ion
Not mentioned in formulation
Water Not tested! 21% Water

Compound Reported Amount mg/mL Actual Formulation
Hydrocortisone 0.12% w/w 1.200 mg/mL 1.316 mg/mL
RRT = 0.99
RRT = 1.01
5.7% area vs parent
4.2% area vs parent
Total:
0.069 mg/mL
0.051 mg/mL
0.120
0.086 mg/mL (Prednisolone)
0.047 mg/mL (Cortisone)
0.133
Unresolved Not reported 0.032 mg/mL (Prednisone)
• Prednisolone and Cortisone cannot be differentiated with existing data
mg/mL calculated using provided formulation density and assuming
comparable response factors
• This assumption may be invalid
Serial No. QCA 58130 Unknown 5 57.50mg
210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 38
AU
0.0
1.0e-1
2.0e-1
3.0e-1
4.0e-1
5.0e-1
6.0e-1
7.0e-1
8.0e-1
9.0e-1
1.0
1.1
1.2
1.3
1.4
30 Unknown 5 5393 (8.987)
244.5612
Post-challenge notes: API and Related Substances
UV Absorbance for Hydrocortisone
UV Absorbance for RRT = 0.99

Post-challenge notes
• Excipient calibration curves (not used for quantitation)
mPEG-350 PEG-400

• All three excipients identified correctly
– PEG mixture identified correctly: 28/72 mixture reported vs. 26/74 actual
• Quantitative accuracy:
– Viscosity of solutions may have effected standard dilutions or injection
accuracy
– Quantitation largely depended on standard concentration due to non-linear
nature of calibration curve
• Detection technique is not suitable for accurate quantitation
– Exponential fit
– MRM quantitation
mPEG-350 PEG-400 PG
Standard Concentration 5.0 mg/mL 2.0 mg/mL 5.0 mg/mL
Actual amount 16.6 mg/mL (49%) 5.8 mg/mL (17%) 2.0 mg/mL (6%)
Reported amount 6.7 mg/mL (20%) 2.7 mg/mL (8%) 2.6 mg/mL (8%)

• Ethanol accuracy: 5.6% reported vs. 7% actual
– Calibration curve not constructed high enough (upper limit = 5%)
– Sample preparation: should have been done in triplicate to obtain %RSD
• Acetaldehyde: not mentioned in formulation
– Acetaldehyde is known Ethanol degradant
• Route: Partial oxidation CH3CH2OH → CH3CHO + H2
• Check a fresh sample preparation
• Interaction with other constituents?
• Buffer accuracy: not mentioned in formulation
– Process or excipient impurity?
– Identity only confirmed by RT (possible coelution)
• Water accuracy: not tested!
– We do not normally quantitate water in sterile DP in ADL
– It would have helped identify a quantitation error in excipients

Conclusions / Future Work
• Surprisingly challenging compared to previous years
• Time Spent
– Here and there for the full five weeks
– One week for one analyst for development / screen / quantitation
– One - two days each for two analysts for API identification / confirmation
• Sample Used
– Two vials left
• Lessons learned
– Check peak purity
– Test for water
– Investigate non-linear calibration
– Investigate alternative MS quantitation

Acknowledgements
• MDO Organizing Committee
• ADL
– Izumi Takagi
– Liz Hewitt
– Travis Anthoine
• BPP
– Mingkun Fu
– Mark Milton

Back-Up Slides

Cortisone
MW = 360.4.
(-2 Da)
Prednisone
MW = 358.4
(-4 Da)
Prednisolone
MW = 360.4
(-2 Da)
Hydrocortisone
MW = 362.4

Serial No. QCA 581
18:33:31
02-Jul-201430 Unknown 5 57.50mg
Time
8.78 8.80 8.82 8.84 8.86 8.88 8.90 8.92 8.94 8.96 8.98 9.00 9.02 9.04 9.06 9.08 9.10 9.12 9.14 9.16 9.18 9.20 9.22 9.24
AU
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
Range: 5.801e+1
8.90
9.09
RRT m/z MW Identity
0.99 405.1 360.4 Cortisone or Prednisolone
Unresolved 403.1 358.4 Prednisone
1.01 405.1 360.4 Cortisone or Prednisolone
Identification of related substances

GC/FID Method Parameters
• Agilent G1888/5890/5973 HSGC/FID/MSD
– Column: DB-624, 30 m ×0.32 mm×1.8 μm
– Oven: 35°C – 240°C, 35 min
– 20 mg/mL sample diluted in DMSO, incubated at 100°C in headspace
sampler for 15 min
• Acetaldehyde confirmation via RT
pA
2.00
4.00
6.00
8.00
10.00
12.00
Minutes
1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50
Ethanol
Methanol
Acetaldehyde Ethanol Standard
DMSO Blank
Acetaldehyde in THF
Unknown sample
Methanol Standard

IC Method Parameters
• Anions
– Eluent: KOH gradient 0.2 – 22 mM, 2.0 mL/min, 30 min
– Column: IonPac AS11 guard (50 mm × 4 mm) and analytical (250 mm ×
4 mm) columns
– Detector: Conductivity mode at 35°C, 1.7% Compensation, 99 mA SRS
• Cations
– Eluent: MSA 20 mM isocratic, 1.0 mL/min, 20 min
– Column: IonPac AS11 guard (50 mm × 4 mm) and analytical (250 mm ×
4 mm) columns
– Detector: Conductivity mode at 35°C, 1.7% Compensation, 59 mA SRS

44 CosMos Method Development Olympics 2014 11Aug2014

Placebo matrix
spiked with
hydrocortisone
Investigational
formulation
*
*
TMS
*

5
Predicted13C

21
212122
5

Mdo2014 takeda

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