1. CoSMoS 2014 Method
Development Olympics
Jeff Kiplinger, Paul Lefebvre, John Tipping, Keith Galyan,
Melissa Grondine, Emma Gatley, Margaret Oti
Averica Discovery Services, Marlborough, MA
2. 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…
3. 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. 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
6. Initial LCMS of Sample
UV 220
TIC
PEG and MPEG
Ghost peaks
“main ingredient”
m/z 363
Related
compounds
7. RICs of Corticosteroids
(“other constituents”)
Prednisone (m/z 359)
Cortisone and Prednisolone
(m/z 361)
UV254
Hydrocortisone (m/z 363)
8. 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
9. 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
10. 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
11. 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
12. 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
13. 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
14. 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
15. 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
