A stabilitv and validation studv of

1%w/w menthol in aqueous &earn

PETER GALLAGHER and SEAN JONES
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In this study an analytical method for the determination of menthol in aqueous
cream was developed, together with the complete validation of the production
zy
z Pharmaceutical
Quality Assurance
Laboratory, St
Thomas’s

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process and stability testing of 18 months’ retained reference samples. Cream
Hospital,
packed in 500ml plastic screw cap containers was tested and studied. Up to now Lambeth Palace
there has only been a small scale (1.40kg) manual manufacture of l%w/w Road, London
menthol in aqueous cream without any formal quality control analysis and SE1 7EH
testing carried out on the product. Peter Gallagher,
BSc, senior

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The purpose of this work was to enable large scale manufacture of the research and
product, which would require development of an analytical method and

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development
validation of the mixing and filling processes. Stability testing of stored reference analyst
Sean Jones, MSc,
iamples was carried out concurrently. MRPharmS, trust
A stability-indicating, gas-liquid chromatographic method, with the use of an quality controller
internal standard, was developed and validated for the product. Stability testing
D f samples stored at 25C i 1C showed no significant degradation over 18 Correspondence:
Mr Gallagher
months. Validation of the manufacturing process by a sequential sampling
technique showed the process to be satisfactory and workable at all stages. lnt J Pharm Pract
The current formulation in use is l%w/w menthol triturated directly into 19975:101-4
iqueous cream BP.
MENTHOL has found widespread use as an ac- bility data were given. A GLC method for an
ive ingredient in pharmaceutical preparations aerosol spray lotion containing menthol, phenol,
In its own and in combination with other drugs benzocaine and pyrilamine maleate has been re-
:or the treatment of respiratory, dermatological ported4 with determination of all components in
md gastrointestinal disorders.’ the lotion mixture showing excellent recovery
It has been shown to have a broad spectrum data.
if pharmacological activity with cooling effects Owing to an increase in demand within the
ind decongestiodantitussive properties in vari- trust for 1 per cent menthol in aqueous cream, a
)us topical, lozenge and nasal spray formula- GLC analytical method was proposed for the
ions.2‘ product with full validation of the mixing and
A detailed review of its efficacy spectrum has filling processes by a sequential sampling tech-
Ieen reported.5 For our purpose, a topical for- nique.
nulation of menthol 1 per cent w/w in aqueous
:ream BP has been developed at the Guy’s & St Aim
rhomas’s Hospital trust mainly for its antipru-
itic activity and cooling effect. It has found some The aim of the work was the development of a
Ise in patients with HIV infection suffering from satisfactory analytical method and production
nild to moderate skin irritation or pruritus due process for menthol 1 per cent w/w in aqueous
o drug side effects. cream, with support from stability work from
Various analytical procedures have been re- reference samples, which would allow the exten-
7orted for quantitation of menthol alone and in sion of the product’s current three-month expiry
he presence of other drugs, but there have been date and improve the overall quality assurance
ew papers on its stability or on analytical meth- of the final product.
ids for menthol alone in a cream base.
A short stability study2 for a 1 per cent men- Materials and methods
hoVS per cent prilocaine hydrogel analgesic for-
nulation has shown the product to be stable for Materials The following materials were pur-
hree months, with further work in progress. A chased: levomenthol crystals BP, l kg (Courtin &
:hromatographic method using gas-liquid chro- Warner Ltd, UK), aqueous cream BP, 500g
natography (GLC) for the simultaneous deter- (Depuy Healthcare, Leeds, UK), camphor BP,
nination of menthol with methyl salicylate in a lOOg (Aldrich Ltd, Gillingham, UK), 95 per cent
opical analgesic has been reported3 but no sta- ethanol BP, 25L (BDH, Poole, UK).

JUNE 1997, THEINTERNATIONALJOURNAL OF PHARMACY

PRACTICE 101
GLC method development All GLC assays were indices were calcu-
conducted using a Varian 3700 series gas chro- lated, based on ra-
matograph, a 10 per cent Carbowax 20M in tios of relative
WHP 80/100 mesh, and a l m column. The car- retention times of
rier gas was nitrogen with a flow rate of menthol and cam-
25ml/min. A flame ionisation detector was used phor and the per-
with the detector temperature set at 220C, the centage of label
injector temperature at 200C, and the column strength.
temperature at 140C. An injection volume of Figure 1 shows
2pL was used, with a run time of approximate- the chromatogram
ly seven minutes. An LA1000 Trivector integra- of menthol stan-
tion system with a compatible IBM computer dard solution with
capture system was used to quantify the menthol retention times of
concentration in the cream. 3.12 and 4.20 min-
For the standard stock solution preparation, a utes for camphor
0.20 per cent w/v stock solution of menthol in and menthol, re-
95 per cent ethanol was prepared fresh daily, spectively.
200mg of menthol reference standard was accu- The method was t

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rately weighed into a lOOml volumetric flask, validated for use;
80ml of 95 per cent ethanol was added and the linearity and preci-
solution made up to 100ml. sion were deter-

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For the preparation of the stock internal stan- mined. A standard
dard solution, 5Omg of camphor reference stan- calibration curve Retention time
dard was accurately weighed into a SOml was constructed by
volumetric flask, 30ml of 95 per cent ethanol (minutes)
linear least square
was added and made up to 5Oml to give a 0.1 regression analysis Figure 1 : Chromatogram for menthol standarc
per cent solution. of peak area ratios and camphor internal standard

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A standard working solution was then pre- of the concentra-
pared by transferring a 5ml aliquot of standard

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stock solution and IOml of camphor internal
standard solution to a 25ml volumetric flask and
making up to 25ml with 95 per cent ethanol.
Sample test solutions were prepared by accu-
rately weighing 5.0g 2 0.lg of sample into a stop-
pered lOOml conical flask and adding
approximately 20ml of 95 per cent ethanol. The
cream was dispersed and melted by heating on a
waterbath at 50C with occasional swirling. The
contents were then carefully transferred to a
tion of the internal standard to the concentratioi
of each of the six calibration standards. The cal
ibration curve is displayed in Figure 2. Calibra
tion standards were prepared by diluting 2, 5, 7
10, 15 and 20ml of menthol stock solution vol
umetrically to SOml to give concentrations o
0.008,0.02,0.028, 0.04,0.06,0.08 per cent w /
menthol, respectively, with a camphor interna
standard concentration of 0.04%.
To confirm that the method was stability in
dicating, a set of three menthol standard stocl

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5Oml volumetric flask via a glass funnel. The solutions were prepared in 95 per cent ethana
conical flask was rinsed with two lOml portions containing 0 . 1 hydrochloric
~ acid, 0 . 1 sodiun
~
of 95 per cent ethanol and transferred to the vol- hydroxide and 6 per cent hydrogen peroxide re
umetric flask, made up to volume with 95 per spectively. Portions of SOml of these treated so
cent ethanol and mixed well. A 30ml portion of
sample solution was filtered using a Whatman
No 5 filter paper. The first 5ml of filtrate was
discarded into another flask.
A 0.04 per cent working sample solution was

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2
then prepared by transferring a lOml aliquot of
sample filtrate and lOml of camphor internal .-
0
* 1.5
standard into a 25ml volumetric flask and mak- E
ing up to 25ml with 95 per cent ethanol. m
It was found that one excipient from the cream I
m 1
was well retained and had a long retention time Y
m
of approximately 30 minutes. To enable its elu- Q)
a 0.5

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tion, a 20-minute interval was inserted into the
method between each set of four sample injec-

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0
tions. This interval could be be extended until the 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08
excipient was eluted and did not show any in- Concentration %w/w
terference.
Peak area ratios for standard and samples Figure 2: Assay of 1 %w/w menthol in aqueous
were calculated. Peak area variation between cream. Plot of ratio of peak areas of menthol ana
two injections was ~ 1 . 5per cent. Identification camphor against concentration of menthol

102 THEINTERNATIONAL JOURNAL OF PHARMACY PRACTICE, JUNE 1997,

 zyxwvu
lutions were transferred to lOOml conical flasks Two samples from each reference were assayed,
and stressed by heating on a water bath at 50C using the GLC method, and the average per-
for three hours. After this time they were allowed centage of label strength and standard deviation
to cool to room temperature. A 5ml aliquot of were calculated. Assays were subsequently re-
each solution was transferred to a 25ml volu- peated following the change to the automated

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metric flask, lOml of the camphor internal stan- mixing process.
dard added and made up to 25ml with 95 per

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cent ethanol. Results and discussion
The solutions were analysed using the GLC
method and compared with an untreated stan- GLC method development The calibration curve
dard working solution. was linear over the range 0.008-0.08 per cent
w/v. The calculated regression coefficient “r”,
Physical testing - A physical examination of slope “c”, and intercept “m”, were 0.99994,
stored reference samples by visual inspection 25.46 and 0.0067, respectively. The precision be-
showed no signs of cracking or phase separation tween a set of 10 standard solutions prepared

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of the cream in any way. from a stock solution had a stan’dard deviation
The appearance, colour, odour and texture of of 0.005 per cent and a coefficient of variation
the cream remained the same throughout the 18- of 0.45 per cent. Confidence limits (95 per cent)
month storage time. were 1.025*0.003.

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Results showed that the menthol did not un-
Method of manufacture of 1 per cent w/w men- dergo any degradation; there were no additional
thol in aqueous cream The manufacture of the peaks under these acidic, alkaline and oxidising
:ream mix was carried out in the St John’s conditions.
:reams and ointments manufacturing suite in the Degradation (70 per cent) to menthone has
pharmacy department at St Thomas’s hospital. been shown to occur under extreme oxidation
The menthol crystals were initially ground to with hot concentrated chromic acid.6 Under nor-
a fine powder using a mortar and pestle. The re- mal storage conditions, menthone does not oc-
quired weight (87.5g) of ground menthol was cur, showing that menthol is a fairly stable
:ransferred to the centre of a glass slab. A quan- compound in most topical formulations and a
:ity of aqueous cream equal to that of the men- long shelf-life is given for some. proprietary
:hol crystals was mixed with the menthol until a cream formulations, eg, Balmosa cream has a
homogeneous, lump-free mixture was obtained. two- to three-year expiry period.
rrituration was continued by adding a further
:hree portions of aqueous cream until a smooth, Method of manufacture of 1 per cent w/w men-
homogeneous mix was obtained. The remaining thol in aqueous cream Assay results for mixing
Skg of aqueous cream was then weighed out and and filling samples by GLC are given in Table 1.
:ransferred into a lOkg Crypto Peerlass mixer. Mixing samples appeared to reach a steady
The menthol and aqueous cream mix on the slab 100 per cent of label strength after only five to
was then transferred to the mixing bowl. The 10 minutes of mixing; the cream was smooth and
bulk cream was mixed at speed 2 for 30 minutes homogeneous at the end of 30 minutes of mix-
and any pieces at the side of the mixer were ing. Mix 6 and mix 7 have slightly lower assay
scraped back into the bulk using a spatula. Cling results than other mixes, possibly because of
film was used to cover the mixing bowl during evaporation of the menthol or a localised effect
the stirring process. The final mixture was on sampling from the bulk cream in the mixing
:hecked for smoothness, homogeneity and ab- vessel. The results are satisfactory overall and
jence of particles. within the acceptable limits of 90.0 to 110.0 per
To validate the production process, a sequen- cent of label strength. The minor concentration
tial sampling technique was employed: 20g of the variation could also be explained by a day-to-day
mix was taken from the mixing bowl at five- GLC assay variation. A coefficient of variation
minute intervals during the mixing process, and of 1.00 per cent was calculated.
20g samples taken at the beginning and after ev- The filling sample results appeared to be sat-
:ry fifth container up until the last filled sample isfactory, indicating uniform, complete mixing
during the filling process. with a mean assay of 100.5 per cent of label
The batch size was 25 350g containers. All strength for six samples.
samples were carefully withdrawn and labelled A relative standard deviation (RSD) of less
at each stage of the process and assayed in du- than 1.0 per cent between duplicate assay injec-
dicate using the GLC method. tions was calculated.

itability assessment of stored reference samples Stability assessment of stored reference samples
:or stability assessment, stored reference samples Taking all the reference samples from the man-
nanufactured using a manual process were as- ual process into account, the mean assay result

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ayed. Samples had storage times in the range 11- was 105.02 per cent of label strength with a stan-
‘7weeks; mean storage at 25C was 49.10 weeks. dard deviation of 8.61 per cent. The results are ,
JUNE 1997, THEINTERNATIONAL
JOURNAL OF PHARMACY
PRACTICE 103
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Table 1: Assay figures obtained during- production
_ Table 2: Assay figures of l%w/w menthol in aqueous
mixing/fiIIing proCess cream. manual Diocess
Batch Storage time Mean menthol 1. Eccles R. Int
Sample Time of sampling
- - Mean menthol
number (min) concentration number (weeks) concentration Pharm J,
1994;8(Suppl
~~
%LS SD BXW0011 %LS SD 11): 17-21
Mix 1 0 7.00 0 016 77 104.0 0.30 2. Nortier YLM,
Mix 2 5 100.00 0.20 01 7 76 99.5 1.30 Van de Haven JA,
Mix 3 10 105.00 0.20 018 75 105.0 0.10 Koks CRW,
Mix 4 15 105.00 0.70 20 75 92.0 0.50 Beijnen JH. Pharm
Mix 5 20 103.00 0.20 21 75 122.0 1.80 World Sci
Mix 6 25 97.50 0.10 22 64 96.0 1.oo 1995;17:214-7.
Mix 7 30 95.80 0.20 23 64 106.0 0.40 3. Sapio J.P,
FF 101.00 0.30 24 60 100.0 0.30 Sethachutkal. K,
F5 101.00 2.00 25 48 99.0 1.oo Moody J.E, J
F10 103.00 0.40 26 48 130.0 0.10 Pharm Sci
F15 101.00 0.90 27 46 113.0 1.30 1979;68:506-8.
F20 98.00 0.70 28 46 111.0 0.80 4. De Fabrizio F.
LF 99.00 0.50 29 45 100.0 0.40 Ibid.
30 41 112.0 0.60 1981;70: 1151-2.
LS = label strength; mix = mixing samples; F = filling samples; FF 31 38 101.0 0.70 5. Eccles R. J
= first filled; LF = last filled; FS = fifth filled container, etc 32 38 103.0 0.20 Pharm Pharmacol
33 36 101.0 0.90 1994;46:618-20.
shown in Table 2. There was some variation 34 36 108.0 0.10 6. Finar 1L.
ranging from 92.0 to 130.0 per cent of label 35 30 97.0 2.50 Organic chemistry
37 26 101.0 0.10 (5th ed), vol 2.
strength which may have been caused by poor 38 26 106.0 1.60 London: Longman
hand mixing of the ground menthol into the 43 11 104.0 0.20 Scientific and
aqueous cream on a slab. However, results were Technical Ltd,
similar to initial values (time to). This manual 1975:379-82.
7. Ortiz-Boyer F,
mixing technique was employed for small quan- Table 3: Menthol assay results obtained from new Tena MT, De
tity specials of around l k g and occasional ex- mixer manufacturing process Castro L,
temporaneous dispensing. The results tend to Batch Date of Number of Initial Valcarcel M. J
indicate a manual operator problem as there is number manufacture samples menthol Pharm Biomed Sci
no evidence of degradation or decomposition BXW001I %LS 1995~13: 1297-
303.

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peaks or any major reduction or increase in ac- 56 09/04/96 2 101.10
8. Kaffenberger
tive peak areas in the chromatograms of any 57 05/06/96 2 101.04 RM, Doyle MJ. J
sample analysed by this GLC method. 58 13/06/96 2 100.40 Chromatogr
Table 3 gives results from the new mixer man- 59 7/10/96 2 104.30 1990;527:59-66.
9. British
ufacturing process. Results are more consistent 60 2211 1/96 2 100.15 Pharmacopoeia
and reproducible overall with all results thus far 61 2 1102197 2 99.20 1993, vol 2.
falling within the range 95.0 to 105.0 per cent of London: HM
label strength. The mean concentration was Figures given show initial values (not stability samples) Stationery Office,
101.00 per cent of label strength with a standard 1993:1001.
10. United States
deviation of 1.63 per cent. Conclusion Pharmacopoeia
This indicates the efficiency and importance of 1990, vol XXII.
a mixer in the final trituration and work-up of A method for the analysis and production of Washington: US
menthol into aqueous cream, producing a supe- per cent w/w menthol in aqueous cream has beel Pharmacopeial
Convention Inc,
rior manufacturing technique than manual tritu- validated for an 8kg batch size. The stability re 1332.
ration alone. sults have shown that the product is stable 01
storage at room temperature for 18 months; ai
Taking into account all the Tables of results, expiry period of one year has been allocated tc
it is seen that most results fell within the range the product. Further work is planned to asses
of 90.0 to 110.0 per cent of label strength, sug- the stability of reference samples manufacture1
gesting that an expiry date of one year would ap- by the mixer process once a sufficient number o
pear to be acceptable for this product. samples have been retained.

104 THE~NTERNATlONALJOURNAL OF PHARMACY PRACTICE, JUNE 1991