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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;

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
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.

Fig. 1. Chemical structures of active, preservatives and degradation product

Standard Preparation
tailed and poor baseline resolution be- thing Sussex, UK). All solvents and re-
Standard solutions consisted of hydro-
tween the propyl p-hydroxybenzoate and agents were HPLC or analytical grade
cortisone acetate (200 lg mL)1), hydro-
hydrocortisone acetate (internal stan- respectively and were obtained from
cortisone alcohol (4 lg mL)1), methyl
dard) was observed. Sample preparation VWR international Ltd (Lutterworth,
p-hydroxybenzoate (2 lg mL)1) and
involved extraction into methanol with Leicestershire, UK), except for de-ionised
propyl p-hydroxybenzaote (0.2 g mL)1)
the aid of heating and vortexing. water which was available in house,
in methanol.
Solich et al. [5] reported a reversed from an Elga PURELAB system (High
phase HPLC method separating all ac- Wycombe, Buckinghamshire, UK).
tives using a Supelco Discovery C18 col- Whatman PuradiscTM 0.45 lm nylon
umn with a mobile phase containing membrane disposable filters were ob- Sample Preparation
methanol, acetonitrile and water. Speci- tained from VWR international Ltd
ficity with regards to p-hydroxybenzoic (Lutterworth, Leicestershire, UK). Zor- A 5 g portion was accurately weighed
acid was not provided. Initial investiga- bax SB-Phenyl guard column (12.5 mm into a volumetric flask (250 mL) and
tions indicated baseline resolution could  4.6 mm I.D., 5 lm), Zorbax SB-Phenyl dissolved in methanol (100 mL) with the
not be achieved between the propyl column (150 mm  4.6 mm I.D., 5 lm) aid of ultrasonication (3 min). If required
p-hydroxybenzoate and hydrocortisone and Vydac Protein & Peptide column the mixture is allowed to cool before final
acetate. Sample preparation was simple (250 mm  4.6 mm I.D., 5 lm) were dilution to volume with methanol. The
and consisted of extraction into acetoni- purchased from Hichrom (Reading, solution was filtered through a Whatman
trile containing an internal standard Berkshire, UK). Hypersil Duet Cation PuradiscTM 0.45 lm nylon membrane
(dexamethasone) followed by centrifuga- (100 mm  4.6 mm I.D., 5 lm), Supelco filter unit prior to injection onto the
tion. Discovery HS F5-3 (100 mm  4 mm HPLC column.
In this paper we describe the devel- I.D., 3 lm) and Develosil High Carbon
opment and validation of a robust, sta- Loading (100 mm  4.6 mm I.D., 5 lm)
bility-indicating method capable of columns were obtained from Hypersil- Results and Discussion
separating hydrocortisone acetate, pre- Keystone (Runcorn, Cheshire, UK), Sig-
servatives and their respective degrada- ma-Aldrich Company Ltd (Poole, Dor- Method Development
tion products in under 15 min, with set, UK) and Phenomenex (Macclesfield,
minimal sample preparation. Cheshire, UK) respectively. Our primary goal was to develop a simple
isocratic, selective separation procedure
for components with diverse chemical
Experimental Apparatus and structures (Fig. 1); however, the task was
Chromatographic Conditions made difficult by the significant differ-
Materials ences in concentration of actives, ranging
The chromatographic system consisted of from 0.01% w/w to 10% w/w, within the
Hydrocortisone acetate, hydrocortisone a Waters Alliance 2960 HPLC system pharmaceutical preparation.
alcohol, methyl p-hydroxybenzoate and coupled to a Waters Alliance 2487 dual Five columns were screened based
propyl p-hydroxybenzoate reference wavelength absorbance detector (Waters upon carbon loading, stationary phase
standards (purity greater than 99.0%) Corporation, Milford, MA, USA) inte- chemistry, particle size, column length
obtained from GlaxoSmithKline (Wor- gration was performed using Beckman and pore size. Selectivity, resolution and

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