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

6

Full-Scan Mass Spectrum

7

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

9

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

12

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

14

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

17

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

19

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

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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

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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

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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

30

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