CoSMoS 2014 Method

Development OlympicsJeff Kiplinger, Paul Lefebvre, John Tipping, Keith Galyan,

Melissa Grondine, Emma Gatley, Margaret Oti

Averica Discovery Services, Marlborough, MA

Summary

• Active identified as cortisol (hydrocortisone)

• Quantitated by HPLC at 1.5 mg/mL

– ID confirmed by co-injection with standard and NMR

• Excipients

– PEG and PEG-Me ether (400-550 avg. MW)

– Ethanol

– Propylene glycol

• Assumptions, assumptions…

Instructions from Ken

• “The sample is a low dose nasal formulation containing

one main ingredient and other constituents… As a bonus

challenge, we would like you to identify other

constituents in the formulation and quantitate them if

possible.”

– We concluded: “one active ingredient, several excipients. The

bonus must be for quantitation of excipients.”

• Sample coming from Catalent…

– We concluded: “probably a nasal spray product in development,

likely a corticosteroid or vasoconstricting decongestant.”

4

CoSMoS 2014 Method Development Olympics Sample

Analytes

Component Concentration (mg/mL)

Hydrocortisone 1.316

Cortisone 0.047

Prednisone 0.032

Prednisolone 0.086

Measured Sample Density = 1.007 g/cm3 at ~21 °C

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CoSMoS 2014 Method Development Olympics Sample

Formulation

Component Concentration (weight %)

Ethanol 7

MPEG

(Methoxy Polyethylene Glycol)

49

Proplylene Glycol 6

Peg 400

(Polyethylene Glycol MW 380-420 Da)

17

Water 21

Initial LCMS of Sample

UV 220

TIC

PEG and MPEG

Ghost peaks

“main ingredient”

m/z 363Related

compounds

RICs of Corticosteroids

(“other constituents”)

Prednisone (m/z 359)

Cortisone and Prednisolone

(m/z 361)

UV254

Hydrocortisone (m/z 363)

Hydrocortisone Determination

• Baseline data

– HPLC, MS, ELSD, PDA-UV

– Use literature and sample description (and our assumptions!) to identify

likely candidates

– MS fragments and NMR support cortisol hypothesis

• Acquire standard to confirm the identity by co-injection

• Quantitation using HPLC-UV against standard sample

– Generic RP gradient, UV 254, 1:10 dilution in MP, 3 point curve

– Calculated concentration of 1.49 mg/mL of crude (vs. 1.32 expected)

– Higher than expected due to prednisone co-elution

Excipients

• Propylene glycol and ethanol seen in GC

– Confirmed by NIST database search

• PEG and MPEG identified from LCMS profiles

– MW distributions from comparison with PEG 400 and MPEG 550

standards

– Quantitation by HPLC using standards

• Quantitative Ion Chromatography identified no

salts/buffers

• Water assumed by use of PEGs

Ion Chromatography - Buffers

• ELSD-based quantitative,

ion-specific assay method

• Routinely used for

assessment of salt form &

contamination of contract

samples

• No ionic material detected

in sample – nothing

retained beyond solvent

front

Quantitation of PG & EtOH

• PG and EtOH identified by direct

injection GCMS and library match

• PG quantitation by direct injection

– EtOH had matrix interference

• Matrix EtOH signal negligible in

headspace GC/FID

• Ethanol quantitation by

headspace GC with 1:10 sample

dilution

• PG determined at 4.8% w/v vs.

expected 6% w/w

• EtOH determined at 10.8% v/v,

expected 7% w/w

Headspace GC/FID of

sample overlaid with

matrix blank

Ethanol,

methanol,

water

Propylene Glycol

(PG)

Low MW

PEGs

Direct Injection GC/MS

PEG / MPEG

• LCMS data clearly

shows PEG + MPEG

signature

• Comparison with

available standards

– PEG 400 is similar

– MPEG 550 is not

Sample

PEG 400

MPEG 550

Quantitation of PEG and MPEG

• Generic RP-LC gradient, SIM on “major peak ions”, 3-point

calibration

– PEG m/z = 371.3

– MPEG m/z = 297.1

• Results

– MPEG = 46% v/v (expected 49% w/w)

– PEG = 6.5 % v/v (expected 17% w/w)

• Issues with our method for PEG quantitation - “picking one peak”

won’t work if standard doesn’t match

Review / Conclusions

• Starting with best guess is useful most of the time

– Easy ID of cortisol, PEG/MPEG, water, ethanol

• Assumptions can become dominant thinking

– Early presentation of a solution narrows thinking

– Opinions/influence of management

– Ignoring unwanted information

• We might have asked more questions

• Excipient quantitation – e.g. to reverse-engineer a formulation – is

difficult

– Variation in source & spec of raw ingredients, cosolvents, salts

Contact Averica

Averica Discovery Services

260 Cedar Hill Street

Marlborough MA 01752

www.avericadiscovery.com

Jeffrey Kiplinger, President

508-757-4600

jeff.kiplinger@avericadiscovery.com