Ž .Pharmaceutica Acta Helvetiae 73 1998 163–165

HPLC determination of the stability of tretinoin in tretinoin–minoxidilsolution

A. Zarghi ), M. Jenabi, A.J. EbrahimianDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Shaheed, Beheshti UniÕersity of Medical Sciences, Tehran, Iran

Received 6 January 1998; revised 13 March 1998; accepted 18 March 1998

Abstract

A new formulation of topical tretinoin–minoxidil solution was prepared and the chemical stability of tretinoin was studied for 6Ž .months at 48C. A reversed phase high performance liquid chromatography HPLC method was developed for determination of tretinoin

using cyanocobalamine as an internal standard. Tretinoin was shown that to be stable for at least 6 months used refrigerated storageconditions. q 1998 Published by Elsevier Science B.V. All rights reserved.

Keywords: Tretinoin; Minoxidil; Solution; HPLC assay; Stability

Ž .Tretinoin all-trans retinoic acid; vitamin A acid hasbeen widely used in the local treatment of several skin

Ž .disorders Boyd, 1989 . It is also known to be effective inthe treatment of male-pattern baldness when coadminis-

Žtered with topical minoxidil solution London-Wong and.Hart, 1990 . It was reported that the precutaneous minoxi-

dil absorption was enhanced by tretinoin as a result ofŽ .increased stratum corneum permeability Ferry et al., 1990 .

We have formulated a tretinoin 0.025%–minoxidil 0.5%Žsolution used the following components: tretinoin Roche,

. Ž .Switzerland 25 mg; minoxidil Alchymarz, Italy 0.5 g;Ž .Propylene glycol BASF, Germany 38 g; Ethyl alcohol

Ž .Merck, Germany 61.5 g; Butylated hydroxy anisoleŽ .Kodak, USA 20 mg. The components were mixed undernitrogen and aseptic conditions. Then the prepared solutionwas packed.

) Corresponding author.

The determination of tretinoin was based on reversedŽphase HPLC method using by cyanocobalamine Roche,

.Switzerland as internal standard. We propose a simple andsensitive method for quantification of tretinoin intretinoin–minoxidil solution.

The operating conditions were as follows. The WatersŽ .HPLC system Waters, Milford, USA employed consisted

of a model 510 pump, a model U6K injector, a model 746recorder and a model 486 UV detector.

The separation was performed on an analytical 150=3.9Žmm i.d. reversed phase Nova pack C 4 mm, particle18

.size column. The wavelength was set at 365 nm. TheŽmobile phase was a mixture of methanol–water 80:20,

.vrv at a flow rate of 1.3 mlrmin.A calibration curve was constructed daily by appropri-

ate dilution of a stock solution of tretinoin with mobilephase containing 1 mgrml cyanocobalamine as internalstandard. The concentration range was 1–8 mgrml. Cali-bration curves were generated by making triplicate 20-mlinjections of the standard concentration. Average peak area

0031-6865r98r$19.00 q 1998 Published by Elsevier Science B.V. All rights reserved.Ž .PII S0031-6865 98 00014-4

( )A. Zarghi et al.rPharmaceutica Acta HelÕetiae 73 1998 163–165164

Fig. 1. Calibration curve of tretinoin.

ratios of tretinoin and internal standard was plotted againstŽ .tretinoin concentration Fig. 1 .

Validation of HPLC assay: The relationship betweenpeak area ratios of tretinoin and internal standard withtretinoin concentration over the concentration was linear

Table 1Reproducibility of the tretinoin analysis

Mean tretinoin Standard deviation Standard deviationŽ . Ž . Ž .mgrml mgrml %

3.09 0.11 3.555.69 0.28 5.067.41 0.69 9.31

Ž .Fig. 2. Chromatograms of A fresh sample of tretinoin–minoxidil solu-Ž . Ž .tion, B 6-month-old sample kept at 48C, C solution containing tretinoin

Žand minoxidil spiked with isotretinoin. 1scyanocobalamine, 2s.minoxidil, 3-tretinoin and 4s isotretinoin .

and the corresponding regression equation was: ysŽ .5.035xq0.593 rs0.9994, ns10 . The HPLC assay

Žwas reproducible and the results of the analysis eight.replicate analysis of each on 3 consecutive days are

represented in Table 1.Stability studies: tretinoin–minoxidil solution was stored

in a refrigerator at 48C. Triplicate samples of solution wereremoved for analysis after intervals of 1,3 and 6 monthsstorage. Also one sample was taken immediately after

Ž .preparation to give the initial ts0 assay value. Therewas no loss of tretinoin from the solution preparation

Ž .stored at 48C over 6 months Table 2 . Results indicatedthat the present formulation can be stored for at least 6months without degradation at 48C. pH was determined in

Ž . Žthe solution 50 ml of each with a pH meter Mettler.Delta 340, Switzerland, sensitivity 0.01 pH unit . The

initial pH was tested as well as after 6 months storage.

Table 2Assay of tretinoin at 48C

Ž . Ž . Ž .Storage time month Tretinoin concentration mgrml Tretinoin as % of initial concentration Standard deviation %

0 250 100 2.41 257 103 2.63 255 102 3.16 242 97 4.2

pH ÕalueInitial 6.86 months 6.75

The average of three experiments is shown.

( )A. Zarghi et al.rPharmaceutica Acta HelÕetiae 73 1998 163–165 165

Results showed that the pH values of tretinoin–minoxidilŽ .solution had not changed Table 2 .

Typical chromatograms of tretinoin are illustrated inFig. 2. Under the chromatographic conditions described,tretinoin, minoxidil and internal standard peaks were wellresolved. In addition in this method tretinoin peak wasseparated efficiently from isotretinoin, therefore been usedfor the kinetic study of tretinoin.

References

Boyd, A.S., 1989. An overview of the retinoids. Am. J. Med. 86,568–574.

Ferry, J.J., Forbes, K.K., Vanderlugt, J.T., Szpunar, G.J., 1990. Influenceof tretinoin on the precutaneous absorption of minoxidil from anaqueous topical solution. Clin. Pharmacol. Ther. 47, 439–446.

London-Wong, D.M., Hart, L.L., 1990. Minoxidil with tretinoin inbaldness. DICP Ann. Pharmacother. 24, 43–44.