Optimisation of a Selective Liquid

Chromatography Procedure for

Hydrocortisone Acetate, Hydrocortisone
Alcohol and Preservatives in a
Pharmaceutical Emulsion 2005, 61, 555–559

V. Chauhan&, B. Conway
GlaxoSmithkline, Consumer Healthcare, Innovation Centre, St Georges Avenue, Weybridge, KT13 0DE, UK;
E-Mail: vijeash.k.chauhan@gsk.com

Received: 15 November 2004 / Revised: 17 March 2005 / Accepted: 4 April 2005

Online publication: 27 May 2005

allow this degradant to be identified and

separated from all other actives.
Abstract A limited number of analytical
An accurate, reproducible and specific stability-indicating method for the high performance methods were found in the literature
liquid chromatography (HPLC) assay of hydrocortisone acetate, hydrocortisone alcohol, methyl that separated some or all of the actives
p-hydroxybenzoate and propyl p-hydroxybenzoate in a pharmaceutical suspension is de- in various matrices, including creams,
scribed. An investigation of several column phases was undertaken and a Zorbax SB-Phenyl solutions and tablets [2–5]. Methods
column gave the best selectivity and specificity due to the p-p interactions between the analytes found in the literature were either com-
and stationary phase. All the components were fully resolved in less than 15 min under isocratic plex in terms of sample preparation,
conditions using UV detection at 254 nm with a water-methanol mobile phase. The stability- used older column technologies or were
indicating method was validated over the linearity range of 25% to 150% of the nominal con- not specific.
centrations of each analyte. Nominal concentrations were hydrocortisone acetate (10% w/w), Lea et al. [2] described normal and
hydrocortisone alcohol (0.2% w/w with respect to hydrocortisone acetate), methyl p-hydrox- reversed phase HPLC methods for oint-
ybenzoate (0.1% w/w) and propyl p-hydroxybenzoate (0.01% w/w) respectively. ments and creams containing hydrocor-
tisone acetate. Separation was limited to
hydrocortisone acetate with a complex
sample preparation step using chloro-
Keywords form as extraction solvent.
Column liquid chromatography Cavrini et al. [3] used a UV spectro-
Hydrocortisone acetate and alcohol photometric method with a solid phase
p-p Interactions extraction (SPE) clean up to measure
Preservatives hydrocortisone acetate and other actives
Pharmaceutical formulations in pharmaceutical creams; however, the
preservatives were removed during the
SPE and not measured.
Wanwimoliuk et al. [4] described an
isocratic HPLC method, which separated
Introduction alcohol maybe present. Various regula- all actives except for the p-hydroxyben-
tory authorities now require the level of zoic acid. The method utilised reversed
Hydrocortisone is classified as a cortico- the degradant to be accurately quanti- phase HPLC with a Nucleosil C18 ana-
steroid drug which is used primarily for fied. lytical column and mobile phase
the treatment of palliative anti-inflam- This pharmaceutical preparation is in consisting of acetonitrile-methanol-water
matory disorders in topical preparations the form of an emulsion containing two (5:55:40 v/v/v) with UV detection at
and as an oral replacement therapy in preservatives, methyl p-hydroxybenzoate 240 nm. The order of elution was methyl
adrenal deficiencies (e.g. Addison’s (0.1% w/w) and propyl p-hydroxybenzo- p-hydroxybenzoate, hydrocortisone alco-
disease). Although the pharmaceutical ate (0.01% w/w). The preservatives can hol, propyl p-hydroxybenzoate and
preparation contains hydrocortisone degrade to p-hydroxybenzoic acid [1] and hydrocortisone acetate. All components
acetate as the main active (10% w/w), the high performance liquid chromatog- were observed within an 8 min window,
its degradation product hydrocortisone raphy assay method must be specific to however, methyl p-hydroxybenzoate

Original Chromatographia 2005, 61, June (No. 11/12) 555

DOI: 10.1365/s10337-005-0555-2
0009-5893/05/06 Ó 2005 Friedr. Vieweg & Sohn/GWV Fachverlage GmbH

Fig. 1. Chemical structures of active, preservatives and degradation product
tailed and poor baseline resolution be-
tween the propyl p-hydroxybenzoate and
hydrocortisone acetate (internal stan-
dard) was observed. Sample preparation
involved extraction into methanol with
the aid of heating and vortexing.
Solich et al. [5] reported a reversed
phase HPLC method separating all ac-
tives using a Supelco Discovery C18 col-
umn with a mobile phase containing
methanol, acetonitrile and water. Speci-
ficity with regards to p-hydroxybenzoic
acid was not provided. Initial investiga-
tions indicated baseline resolution could
not be achieved between the propyl
p-hydroxybenzoate and hydrocortisone
acetate. Sample preparation was simple
and consisted of extraction into acetoni-
trile containing an internal standard
(dexamethasone) followed by centrifuga-
tion.
In this paper we describe the devel-
opment and validation of a robust, sta-
bility-indicating method capable of
separating hydrocortisone acetate, pre-
servatives and their respective degrada-
tion products in under 15 min, with
minimal sample preparation.
Experimental
Materials
Hydrocortisone acetate, hydrocortisone
alcohol, methyl p-hydroxybenzoate and
propyl p-hydroxybenzoate reference
standards (purity greater than 99.0%)
obtained from GlaxoSmithKline (Wor-
556
thing Sussex, UK). All solvents and re-
agents were HPLC or analytical grade
respectively and were obtained from
VWR international Ltd (Lutterworth,
Leicestershire, UK), except for de-ionised
water which was available in house,
from an Elga PURELAB system (High
Wycombe, Buckinghamshire, UK).
Whatman PuradiscTM 0.45 lm nylon
membrane disposable filters were ob-
tained from VWR international Ltd
(Lutterworth, Leicestershire, UK). Zor-
bax SB-Phenyl guard column (12.5 mm
 4.6 mm I.D., 5 lm), Zorbax SB-Phenyl
column (150 mm  4.6 mm I.D., 5 lm)
and Vydac Protein & Peptide column
(250 mm  4.6 mm I.D., 5 lm) were
purchased from Hichrom (Reading,
Berkshire, UK). Hypersil Duet Cation
(100 mm  4.6 mm I.D., 5 lm), Supelco
Discovery HS F5-3 (100 mm  4 mm
I.D., 3 lm) and Develosil High Carbon
Loading (100 mm  4.6 mm I.D., 5 lm)
columns were obtained from Hypersil-
Keystone (Runcorn, Cheshire, UK), Sig-
ma-Aldrich Company Ltd (Poole, Dor-
set, UK) and Phenomenex (Macclesfield,
Cheshire, UK) respectively.
Apparatus and
Chromatographic Conditions
The chromatographic system consisted of
a Waters Alliance 2960 HPLC system
coupled to a Waters Alliance 2487 dual
wavelength absorbance detector (Waters
Corporation, Milford, MA, USA) inte-
gration was performed using Beckman
Chromatographia 2005, 61, June (No. 11/12)
PeakProÒ (Beckman Coulter Ltd, High
Wycombe, Buckinghamshire, UK) Chro-
matography Data System. Specificity
testing was achieved using a Agilent 1100
series diode array detector (Agilent
Technologies, Palo Alto, California,
USA). Analysis was carried out using
reversed phase chromatography on a
Zorbax SB-Phenyl column (150 mm 
4.6 mm I.D., 5 lm, 12.5 mm 4.6 mm I.D.
guard) with a mobile phase consisting of
a mixture of methanol–water (60:40 v/v)
at 35 °C. An injection volume of 10 lL
(for the analysis of Hydrocortisone ace-
tate only) or 100 lL (for the analysis of
preservatives and hydrocortisone alco-
hol) with a detection wavelength of
254 nm were used.
Standard Preparation
Standard solutions consisted of hydro-
cortisone acetate (200 lg mL)1), hydro-
cortisone alcohol (4 lg mL)1), methyl
p-hydroxybenzoate (2 lg mL)1) and
propyl p-hydroxybenzaote (0.2 g mL)1)
in methanol.
Sample Preparation
A 5 g portion was accurately weighed
into a volumetric flask (250 mL) and
dissolved in methanol (100 mL) with the
aid of ultrasonication (3 min). If required
the mixture is allowed to cool before final
dilution to volume with methanol. The
solution was filtered through a Whatman
PuradiscTM 0.45 lm nylon membrane
filter unit prior to injection onto the
HPLC column.
Results and Discussion
Method Development
Our primary goal was to develop a simple
isocratic, selective separation procedure
for components with diverse chemical
structures (Fig. 1); however, the task was
made difficult by the significant differ-
ences in concentration of actives, ranging
from 0.01% w/w to 10% w/w, within the
pharmaceutical preparation.
Five columns were screened based
upon carbon loading, stationary phase
chemistry, particle size, column length
and pore size. Selectivity, resolution and
Original