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 　　 11Aug2014

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.CosMos Method Development Olympics 2014 　　 11Aug2014

• Why MDO?• Cross-functional collaboration• Interesting way to participate

with the scientific community• I wanted to come to CoSMoS• Fun

Strategy

Plan:

1. Identificationa) Assess

b) Obtain standards

c) Confirm

2. Assay developmenta) Choose appropriate

instrumentation / detector

b) pH screen

c) Stationary phase screen

3. Identify and quantitate other constituentsa) IC screen for salts

b) GC screen for volatiles

Goals:

4. Primary: solve the challengea) Use existing methods

b) Instruments available:• UPLC/TQD, UPLC/SQD,

UPLC/HRMS , GC/MS, GC/qTOF, FTIR/Raman/nIR, NMR

5. Secondarya) Other constituents

b) Method assessment

Challenges:

6. Sample Type

7. Sample Information

8. Time!


Outline







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 scanCosMos Method Development Olympics 2014 　　 11Aug2014

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.edu/staff/kind/Metabolomics/MS-Adduct-Calculator

Table 1. Monoisotopic exact masses of molecular ion adducts often observed in ESI mass spectra Your M here:

362.30000Ion name Ion mass Result:1. Positive ion mode M+3H M/3 + 1.007276 121.773943M+2H+Na M/3 + 8.334590 129.101257M+H+2Na M/3 + 15.7661904 136.532857M+3Na M/3 + 22.989218 143.755885M+2H M/2 + 1.007276 182.157276M+H+NH4 M/2 + 9.520550 190.670550M+H+Na M/2 + 11.998247 193.148247M+H+K M/2 + 19.985217 201.135217M+ACN+2H M/2 + 21.520550 202.670550M+2Na M/2 + 22.989218 204.139218M+2ACN+2H M/2 + 42.033823 223.183823M+3ACN+2H M/2 + 62.547097 243.697097M+H M + 1.007276 363.307276M+NH4 M + 18.033823 380.333823M+Na M + 22.989218 385.289218M+CH3OH+H M + 33.033489 395.333489M+K M + 38.963158 401.263158M+ACN+H M + 42.033823 404.333823M+2Na-H M + 44.971160 407.271160M+IsoProp+H M + 61.06534 423.365340M+ACN+Na M + 64.015765 426.315765M+2K+H M + 76.919040 439.219040M+DMSO+H M + 79.02122 441.321220M+2ACN+H M + 83.060370 445.360370M+IsoProp+Na+H M + 84.05511 446.3551102M+H 2M + 1.007276 725.6072762M+NH4 2M + 18.033823 742.6338232M+Na 2M + 22.989218 747.5892182M+3H2O+2H 2M + 28.02312 752.6231202M+K 2M + 38.963158 763.5631582M+ACN+H 2M + 42.033823 766.6338232M+ACN+Na 2M + 64.015765 788.615765

Your M here:2. Negative ion mode 362.30000Ion name Ion mass Result:M-3H M/3 - 1.007276 119.759391M-2H M/2 - 1.007276 180.142724M-H2O-H M- 19.01839 343.281610M-H M - 1.007276 361.292724M+Na-2H M + 20.974666 383.274666M+Cl M + 34.969402 397.269402M+K-2H M + 36.948606 399.248606M+FA-H M + 44.998201 407.298201M+Hac-H M + 59.013851 421.313851M+Br M + 78.918885 441.218885M+TFA-H M + 112.985586 475.2855862M-H 2M - 1.007276 723.5927242M+FA-H 2M + 44.998201 769.5982012M+Hac-H 2M + 59.013851 783.6138513M-H 3M - 1.007276 1087.907276

Hypothesis: m/z = 362.3

http://fiehnlab.ucdavis.edu/staff/kind/Metabolomics/MS-Adduct-Calculator



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)CosMos Method Development Olympics 2014 　　 11Aug2014

M + H Accuracy (ppm)

1. C19H33O2F2S -0.12. C21H31O5 -0.73. C17H27N4F4 -0.84. C16H31O4F4 2.95. C22H32OFS 3.06. 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: 6.61E5F: FTMS + c ESI d Full ms2 [email protected] [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

Rela

tive A

bundance

327.1968

309.1861

345.2075

267.1750

297.1861

364.2221121.0649249.1645

279.1752

209.1334187.1124169.1015147.0810109.0651

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 unknownCosMos Method Development Olympics 2014 　　 11Aug2014

API Confirmation: 1H NMR


Placebo spiked with hydrocortisone standard

Unknown sample in D2O

-0.20.00.20.40.60.81.01.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.85.05.25.45.65.86.0f1 (ppm)

Outline







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)


UnknownStandard MixBlank

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)CosMos Method Development Olympics 2014 　　 11Aug2014

UnknownStandardBlank

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

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 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00

Eth

ano

l

Pro

pyl

ene

Gly

col

Ace

tald

ehyd

e

BlankUnknown

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

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

Intensity

0

5x108

Minutes1.00 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

Minutes1.00 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


m/z100 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

http://en.wikipedia.org/wiki/File:Triton_X-100.png

http://en.wikipedia.org/wiki/File:Polyethylene_glycol.png

http://en.wikipedia.org/wiki/File:Polysorbate_20.png


• Tween-80

• Placebo

• Triton-X

• Labrasol®

• PEG-400


Time1.00 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

1.73e810.53

10.36

10.00

9.539.228.818.32

7.697.40

10.71 11.7011.8311.93

12.08

12.31

Cosmos_placebo x5_3 1: MS2 ES+ TIC

4.60e85.865.51

4.854.30

3.863.313.08

0.79

0.46

2.982.30

1.521.90

6.28 6.42

6.56

6.5810.416.89 10.25

9.508.157.88 9.268.87 10.64 11.8612.09

Triton-x 2mg 1: MS2 ES+ TIC

1.89e811.2310.649.509.427.587.456.93

6.395.955.454.99

8.988.15 10.13 12.2111.72 12.45

12.71

12.84

13.06

Labrasol 2mg 1: MS2 ES+ TIC

2.73e86.115.82

5.48

5.11

4.684.143.89

3.603.211.050.722.071.96

6.35

6.807.0011.60

7.33 11.2710.6610.289.428.677.74

12.2012.3212.70

PEG400 3 1: MS2 ES+ TIC

4.29e811.636.85

6.226.015.73

5.004.584.06

3.373.24

11.327.0011.147.16

10.987.28

11.8212.08

12.2612.36


• PEG-400 ESI (+) spectra extracted 4 min – 9.5 min overlaid with unknown


m/z150 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

459.3278371.1924

327.1373

283.0260

238.9818

194.9689

213.9570 260.9644

415.2563

392.9949

503.2910

547.3471

591.3284

635.3179

679.3762723.2919

M + H = 283, when n = 6M + H = 327, when n = 7M + H = 371, when n = 8M + H = 415, when n = 9M + H = 459, when n = 10M + 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


m/z150 200 250 300 350 400 450 500 550 600 650 700 750 800

%

0

100

3 mPEG350 2 915 (6.689) Cm (835:947) 3: MS2 ES+ 9.79e6

385.2866

341.2102297.1251

253.0425208.9856

165.0102

186.9116230.9416 275.0502

429.3125473.2740

517.2883

561.3499

605.3434

649.3272710.4086

M + H = 296, when n = 6M + H = 340, when n = 7M + H = 384, when n = 8M + H = 428, when n = 9M + H = 472, when n = 10M + 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


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!

Time8.78 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

30 Unknown 5 5: Diode Array 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

http://en.wikipedia.org/wiki/File:Propylene_glycol_chemical_structure.png

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


m/z100 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

371.1924

194.9689

213.9570

283.0260327.1373

415.2563

459.3278

503.3278

547.3471

591.3284

mPEG m/z selectedPEG 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







Reported Result vs Actual Formulation


Component Reported Formulation Actual Formulation

API 0.12% w/w Hydrocortisone 1.316 mg/mL Hydrocortisone

Related Substances5.7% area vs parent RRT = 0.994.2% area vs parent RRT = 1.01

Cortisone: 0.047 mg/mLPrednisone: 0.032 mg/mLPrednisolone: 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

Volatile0.29% area vs Ethanol Acetaldehyde

Not mentioned in formulation

Buffer140 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 = 0.99RRT = 1.01

5.7% area vs parent4.2% area vs parent

Total:

0.069 mg/mL0.051 mg/mL0.120

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 datamg/mL calculated using provided formulation density and assuming comparable response factors• This assumption may be invalid

nm210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400

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) 5: Diode Array 1.534244.5612

Post-challenge notes: API and Related Substances

UV Absorbance for HydrocortisoneUV 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


Thank you!

Questions?


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


Time8.78 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

30 Unknown 5 5: Diode Array 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

Minutes1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50

Eth

ano

l

Met

hano

l

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


-1.0-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.5f1 (ppm)

Predicted 1H NMR Spectrum 2

IK-Placebo-Unknown-Jun2014.20.fid19-JUNE-14 Sample from Izumi for an analytical comptetition Formulation 10uL in 50 uL total (rest D2O) VERY viscous WATER H2O+D2O {C:\Bruker\TOPSPIN\data} Milton 59 1

26 20

1716''

2318

5 12'29

11''21'8'7'

16' 21''281'

1''12''

8''7''

22 2'' 2' 2711'


-0.20.00.20.40.60.81.01.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.85.05.25.45.65.86.0f1 (ppm)

Placebo matrix spiked with hydrocortisone

Investigational formulation

*

*

TMS*


0.00.51.01.52.02.53.03.54.04.55.05.56.06.5f2 (ppm)

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

f1 (

ppm

)

IK-Placebo-Unknown-Jun2014.28.ser20-JUNE-14 Sample from Izumi for an analytical comptetition 10uL in 50 uL total (rest D2O)

1

2

3

O4

5

6

7

8

9

1011

12

13

14

O15

16

OH17

OH18

19

CH32021

22

OH23

24

25

CH326

H27

H28

H29

5

Pre

dict

ed 13

C


0.00.51.01.52.02.53.03.54.04.55.05.56.06.5f2 (ppm)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

f1 (

ppm

)

IK-Placebo-Unknown-Jun2014.27.ser20-JUNE-14 5mm probe Sample from Izumi for an analytical comptetition Formulation 100uL in 700 uL total (rest D2O) WATER H2O+D2O {C:\Bruker\TOPSPIN\data} Milton 59

1

2

3

O4

5

6

7

8

9

1011

12

13

14

O15

16

OH17

OH18

19

CH32021

22

OH23

24

25

CH326

H27

H28

H29

21

212122

5


mdo2014 takeda

Education