3. 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
5. 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
9. 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
11. 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
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13. Other Components
1) 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.
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15. Other Components
2) 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.
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16. Other Components
3) 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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22. 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.
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24. 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
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26. Achiral UPLC Screen
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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)
27. 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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28. 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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29. Validation Parameters
Parameter 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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32. 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
34. 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”
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36. 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
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