Pharmaceutical Research, Vol. 6, No.5, 1989

Technical Note

In Vitro DiffusionCell Design and Validation.I.A Stability-Indicating High-Performance LiquidChromatographic Assay for Betamethasone 17-Valeratein Purified .Isopropyl Myristate Receptor Phase

Eric w. Smith1,2 and John M. Haigh1

Received September 26, 1988;accepted January 10, 1989KEY WORDS: high-performance liquid chromatography (HPLC); betamethasone 17-valerate; beta-methasone 21-valerate; receptor phase; isopropyl myristate. .

INTRODUCTION

The development of a reliable in vitro permeation sys-tem necessitates the use of a precise and accurate method ofquantifying tb~ amount of permeant partitioning from themembrane into the cell receptor phase. Aqueous donor andreceptor chamber fluids have ~en used in the majority ofreported investigations, which makes quantitative permeantanalysis relatively facile. Alternatively, radiolabelled diffu-sants have been used and flux rates monitored by scintilla-tion counting, obviating the need for chromatographic sep-aration of the receptor-phase components. However, thistechnique is not applicable when nonlabelled compounds orcommercial dosage forms are to be evaluated by a cell sys-tem. Furthermore, several studies indicate that aqueous re-ceptor phases may not present an optimal partitioning envi-ronment for certain lipophilic permeants (1~), thereby im-pairing accurate flux monitoring due to limited diffusantsolubility. Several attempts have thetefore been made to im-prove the partitioning environment within these systems, bythe addition of surfactants for example (4). A lipophilic re-ceptor environment appears beneficial for corticosteroidpartitioning, and thus, the use of isopropyl myristate hasbeen investigated because of its bipolar properties that tendto mimic the biochemical composition of the skin (5,6). Be-tamethasone 17-valerate and its 21-valerate degradationproduct are highly soluble in isopropyl myristate and thisnonaqueous solvent willnot augmentC-17-to-C-21ester deg-radation reactions.

The UV absorbance of the receptor-phase solutions hasbeen measured directly by spectrophotometry without priorchromat9graphic'separation (7-10). However, compoundsother than the diffusant of interest may permeate from thedonor compartment, or may leach fromthe membrane, andmay contribute to this measured absorbance. Therefore,

1 School of Pharmaceutical Sciences, Rhodes University, Grahams-town, 6140 South Africa.

2 To whom correspondence should be addressed.

chromatographic separation of the receptor-phase constitu-ents is considered essential for accurate analytical estima-tion and to distinguish degradation products or other UV-absorbing moieties. Although Poulsen et al. (5) have usedthin-layer chromatography to separate corticosteroids in iso-propyl myristate, high-performance liquid chromatographyappears to be the analytical technique of choice for accuracyand precision. The developed methodology is similar to thatdescribed previously for the assay of betamethasone valer-ate esters in topical formulations (11)and uses a multidimen-sional, column-switching technique for isolation of the drugmoieties and internal standard from the lipid phase, the latterpresenting significantchromatographic problems without ad-equate separation.

MATERIALSAND MEmODS

Reagents and Chemicals

The acetonitrile and methanol were glass distilled andspectral grade (Burdick and Jackson, U.S.A.). The high-performance liquid chromatographic-grade water used in themobile phase was purified by a Milli-Q system (Millipore,Bedford, Mass.), and the mobile phase ftltered through a0.45-ILmmembrane ftlter (Type BD, Millipore). All chemi-cals were analytical reagent grade and were used as re-ceived. The betamethasone 17-valerate was obtained fromGlaxo, South Africa; the norethisterone was from Ethnor,South Africa; and the betamethasone 21-valerate was an au-thentic specimen from the British Pharmacopoeia Commis-sion. The isopropyl myristate (Merck, West Germany) andthe propylene glycol (Saarchem, South Africa) were bothsynthesis grade, rated at 98 and 95% pure, respectively.Samples of higher purity were not available commercially.

Apparatus

The high-performance liquid chromatograph consistedof a solvent delivery system (Model 6000A, Waters,U.S.A.);.a syringe-Io@ng; six-port sample injector (Model

431 0724-8741/89/0500-0431$06.00/0 @ 1989 Plenum Publisbing Corporation

432

7125, Rheodyne, U.S.A.tauiPped with a loop column(MPLC, 3cm x 4.6-nunL , packed with Lichrosorb RP-18,Brownlee, U.S.A.) and a ariable-wavelength UV detector(Model SF769, Kratos Analytical Instruments, U.S.A.) cou-pled to a strip chart recorder (Modell00A, Perkin-Elmer,Palo Alto, Calif.) or an integrator (Model 3390A, Hewlett-Packard, Palo Alto, Catif.). A diode-array UV detector(Model I040A, Hewlett-Packard) was employed in place ofthe variable-wavelength detector for spectral analysis. Theanalytical column(25cm x 4.6-nunLd.) was custom packedwith octadecylsilane (Techsil 10 IJom,HPLC Technology,U.K.). Valve switching and loop column flushing were ac-complished manually.

ChromatographyConditions

The ion-free mobile phase was prepared by adding 45parts by volume of water to 55 parts acetonitrile in a stop-pered flask and allowingthe mixture to equilibrate to roomtemperature prior to simultaneousfIlteringand degassing un-der vacuum. The mobile-phase flow rate was set at 1.5 mIlmin and all chromatographywas carried out at ambient tem-perature with the detector wavelength set at 239 om.

Purification of the IsopropylMyristate

To assess its purity, an aliquot of isopropyl myristatewas extracted with an equal volumeof mobile phase, and 10IJoIof the latter injected into the chromatograph. Large ab-sorption peaks between 4.5 and 6.5 min were presumablydue to impurities in the lipid. An extraction of the isopropylmyristate with mobile phase containing betamethasone 17-valerate and norethisterone in solution (Fig. 1) indicatedthat, while it may be possible to resolve the norethisterone

N IPM

IPM

T4

T7

I . I

5 6TIME / minutes

Fig. 1. Typical chromatogram of an unpurified isopropyl myristateextract showing elution interference of the impurities with the be-tamethasone 17-valeratepeak. B, betamethasone:17~valerate;IPM,impurities in isopropyl myristate; N, norethisterone.

Smith and Haigh

peak from the large impurity band, betamethasone 17-valerate eluted between the two impurity peaks, making ad-equate resolution impossible. Spectral analysis of the impu-rity peaks at 4.95 and 5.88min (Fig. 2) indicates that bothpeaks have a UV absorbance maximum at approximately225 om, implying that these peaks are probably not due toimpurities of steroidal,origin. Similar spectral analysis of the.betamethasone 17-valerateand norethisterone peaks demon-strate the indicative steroidal absorbance maximum at240 om.

Purification of largewolumes of i:;opropyl myristate bysuccessive extractions with either acetonitrile or water wasnot feasible, as the former solubilizedsome of the lipid phasewith each extraction, and the impuritiesdid not partition intothe aqueous phase. Successive extractions of the isopropylmyristate with propylene glycol aliquots significantly dimin-ished the concentrations of impurities, and adequate re-moval for chromatographic sensitivity was effected by 15isovolumetric extractions conducted in large volume sepa-rating flasks with centrifugation of the lipophilic layer. Thismethod was adopted for the purification of all synthesis-grade isopropyl myristate used in subsequent in vitro per-meation experitnents.

Internal Standard Solutions

The internal standard solution was prepared in mobilephase at a norethisterone concentration of 3.5 lLg/ml.Underthe operating conditions described above it was found thatthe chromatographic system adequately resolved the nor-ethisterone (4.2 min) from betamethasone 17-valerate (5.6min) and betamethasone 21-valerate (6.5 min), together withthe preservatives chl~rocresol (3.6 min)and methyl hydroxybenzoate (2.9 min) contaitJ.e'ilin some conunercial topicalformulations evalilated by the in vitro diffusion cell. Reso-lution of the internal standard peak from enhanced solventfronts due to donor solutions of corticosteroid in alcoholicsolution was also achieved.

Calibration Standard Solutions

Standard stock solutions of betamethasone 17-valeratewere prepared in purified isopropyl myristate at concentra-tions of 2.5,5.0, 7.5, 10.0, 15.0,20.0, 25.0, 30.0, 35.0, and40.0 lLg/ml.These solutions were used to test for the linearityof detector response and recovery characteristics of the cor-ticosteroid from the isopropyl myristate. Similarly, in orderto quantify any degradation product, solutions of betameth-asone 21-valerate were prepared in isopropyl myristate atconcentrations of 0.5, 1.0, 1.5, and 2.1 JLg/ml.These rangesrepresented the expected magnitudes of the permeants in thereceptor cell solution.

Sample Preparation

During the course of the permeation experiment, 4O-ILIaliquots were sampled from the calibration standard stocksolutions or were withdrawn from the receptor chamber ofthe diffusion cell at specified time intervals using a fixed-volume transfer pipette (Brand, West Germany). These sam-ples were placed into small glass collection tubes (4 nun-Ld.x 50-nun length), the tubes were covered (paraf1lm, Amer-

HPLC Assay for Betamethasone 17-Yalerate in Isopropyl Myristate

10.0

:::><x:E'-WUz<x:IX:)a::a(I)IX:)<x:

WAVELENGTH / nm

433

IPM

2926.5 6.0 5.5 5.0 4.5

TIME / minutes

Fig. 2. Three-dimensionalspectral plot ofbetamethasone 17-valerate(B), impurities in isopropyl myristate (lPM), and noreth-/ isterone (N).'"

ican Can Co., U.S.A.) and stored under refrigeration (4°C)until assayed. Prior to injection, 40 ILlof internal standardsolution was added to the isopropylmyristate aliquot in eachtube and the mixture vortexed for 30 sec, ensuring a homo-geneous, fine dispersion of the two immiscible phases andthereby facilitating optimal corticosteroid partitioning be-tween the phases. The samples were then centrifuged for 60sec to assist in the separation ofthe phases and 10 ILlof thelower, mobile phase layer was withdrawn by syringe for in-jection into the chromatograph.

Injection Procedure

The sample injection and column-switchingprocedurewas similar to that described previously (11). Lipophiliccomponents trapped on the loop column were forwardflushed to waste using 1 ml methanol followed by 5 ml mo-bile phase, thereby no reequilibration of the loop columnwas required prior to the next injection. It was found thatmethanol washes were adequate for displacing bound li-pophilic components from the loop column, and the disrup-tive effects of using a stronger eluent such as tetrahydrofu-ran (12-14) were, therefore, avoided. Calibration standardsolutions were interspaced between sample injections suchthat each solution was ii:Yectedat least three times for meansto be calculated. Peak height ratios from injected sampleswere compared to the ratios from calibration standard solu-tions to quantify the permeant concentrations using the rel-ative response factor method (15,16).

4.0

RESULTS AND DISCUSSION

Figure 3 depicts chromatographic traces of the isopro-pyl myristate receptor solutions obtained from diffusionrunsusing different, commercial, betamethasone 17-valerate-containing topical formulations (Betnovate, Glaxo, SouthAfrica) as the donor phase and hUmaIJstratum corneum asthe diffusion membrane. A typical chromatogram of the iso-propyl myristate extract from a cream diffusion experimentis shown in Fig. 3a and exemplifies the resolution betweenthe formulation preservative (chlorocresol) and the norethis-terone internal standard. Figure 3b is a chromatogram froma lotion permeation experiment and demonstrates the largeabsorption band generated by the methyl hydroxy benzoatepreservative. Figures 3C~d d show chromatographic tracesfrom ointment and scalp pplication permeation experi-ments, respectively; in these cases there are no preser-vatives in the dosage forms. The presence ofbetamethasone21-valerate fractions was rarely observed during routinechromatography. In instances when the degradation productwas detected during assay of the topical dosage forms priorto initiating the permeation experiment, this moiety was alsoobserved and quantified in the receptor chamber isopropylmyristate.

Specificity and Linearity Studies

To ensure the specificity of the detector response a UYspectrum was recorded at half-second intervals as the inter-nal standard and betamethasone 17-valerateeluted from the

434

N c N MHB N B

N

JB BB

SF

(c)(a) (d)

~TIME

Fig. 3. Typical chromatographic traces from extracts of isopropylmyristate receptor phases from cream (a), lotion (b), ointment (c),and scalp rapplication (d) permeation experiments. B, betametha-sone 17-vaferate;C, chlorocresol;MHB, methyl hydroxy benzoate;N, noretbisterone; SF, alcoholic solvent front.

analytical columnafter sample aliquot injection. These spec-tra were compared to.those obtained after injection of a puresolution of each compound. No obvious differences werenoted, implying that tile steroid peaks obtained from theextract of the lipid receptor solution were not augmented byany other extraneous constituents. Calibration curves (TableI) constructed on the basis of the peak height ratios of beta-methasone 17-valerate/internalstandard versus betametha-sone 17-valerate mass were mrear over the concentrationrange studied (2.5-40.0 ,...g/ml).Similar linearity was ob-tained for the betamethasone 21-valerate calibration curveover the concentration range 0.5-2.1 ,...g/ml,indicating thatthe assay method is suitable for quantitative purposes overwide corticosteroid concentration ranges and is sensitive totrace amounts of the permeants.

a B 17-V, betamethasone 17-valerate;B 21-V, betamethasone 21-valerate; NOR, noretbisterone.

Smith and Haigh .

Precision, Accuracy,and RecoveryStudies

To determine the precision, accuracy, and recovery ofthe method, solutions of betamethasone 17-valerate weremade up in normal internal standard solution at concentra-tions of 5.0, 10.0, 20.0, 30.0, and 40.0 ,...g/ml.The averagepercentage recovery observed over this spike concentrationrange was 99.78% (SD, 1.68%;N = 4 samples at each con-centration), indicating that negligibleloss of betamethasone17-valerate is experienced during sample preparation andthat preferential partitioning of the corticosteroid occursfrom the isopropyl myristate solution into the acetonitrilelwater phase.'fI!e method is, therefore, sufficiently accurateand precise for analytical purposes.

On-lineCleanup

The column-switchingtechnique was found to be highlysuitable and efficient for the on-line cleanup of samples andeffected adequate isolation of the lipophilicfraction from theanalytical system, thereby obviatingchromatographic inter-ference and minimizing sample handling. Furthermore, de-terioration of the analytical column performance was notexperienced even after a year of continuous use.

CONCLUSIONS

The high-performance liquid chromatographic tech-nique developed is highly efficient for the analysis of beta-methasone 17-valerateconcentrations in isopropyl myristateused as the receptor phase of in vitro diffusion cells. Extrac-tion of the drug moiety from the lipid phase is effected by acombination of liquid-liquid extraction and on-line samplecleanup using a switching v~e and loop column assembly.The method is shown to be'relatively simple, accurate, andlinear over the broad permeant concentration range experi-enced in diffusion experiments and has the capability of si-multaneously quantifying degradation products and topicalformulation preservatives in the cell receptor solution. Themajor advantage of having this reliable analytical techniqueavailable is that it allows the accurate monitoring of noma-diolabelled corticosteroid permeation in laboratory diffusionexperiments, thereby allowing the testing of commercialproducts. Slight modifications to the methodology have al-lowed the assay of other corticosteroids, such as hydrocorti-sone, indicating that the technique is applicable to a diverserange of corticoid permeants.

ACKNOWLEDGMENTS

It is with sincere gratitude that grants are acknowledgedfrom the South African Council for Scientific and IndustrialResearch, the Rhodes University Council, and the H. Brad-low Foundation.

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Tle I. Linearity Data and Calibration Curve Constructiona

Internal Slope Intercept CorrelationCompound standard x 1000 x 100 coefficient N

B 17-V NOR 2.680 1.343 0.9992 30B21-V NOR 1.371 . 0.295 0.9958 16

HPLC Assay for Betamethasone 17-Valerate in Isopropyl Myristate

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