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    08 Morais Vit E

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    Internationa Journal of Pharmaceutics 468 (2014) 455-488, rine Content fi lable at ScienceDirect International Journal of Pharmaceutics journal homepage: www.elsevier.com/locat ijpharm Vitamin E@anoemmulsions characterization and analysis Jacqueline M. Morais Diane, J. Burgess * Depraen of Parmele eon of harman vey of Comet Sr, Comma, O26, ed Se (ewes ‘Fakta, ecehed 23 Ores 2012 acete revised for 12 Febery 2014 ‘Acctpted 15 Fbrony 2004 ‘vane ste 19 Feraty 2 “he sims ofthis work were to characterize vitamin canola oll nanoemulsons andto develop apractal [RP-HPLC method for vitanin € acetate estimation inthe canola cll nanoemulsons Currently salable ‘methods to analyze vitamin € in lipid-emulsions are time-consuming and do not allow adequate Separation of vitamin & and its esters. The mnoemulsions were chaacteried for partition coefficient Detween emulsion phases, drug loading fee drug, micellar solubilization, encapsulation efieney and ‘amin & concentration inthe emulsion external aqueous phase 35a function of time, Fomlation stably under stress conditions was aso evaluated. The nanoemulsions were stable during the test period at 25 and 32°C with no signicant change in mean droplet dlamete. The rests sgest that Drolonge release of lipoptilic drugs can be achieved using nancemulsions. The RE-HOLE method developed showed linearity, selectivity ane efficiency To the best of our knowledge this is the fst praticalmethodfor vitamin analysis in ipd-emlsions.Tismethod may alsabe applcdto the analysis Taywort Vissin Ecanca of amoemons iain € senate OKIE sala method fo sitrinE of vitamin and its esters in othe lipid formulations, and for qualty contol purposes 1. Introduction [Nano-and submicron-sized emulsions are becoming increasing important as a delivery vehicle for hydrophobic drugs and other active materials. Nano-sized emulsions have droplets in the size ange of 20 ~ 200nm (Tadros et al, 2004), whereas submicron- sized emulsions have droplet size distribution ranging between 50, {and 1000.m (Tamilvanan, 2004) tis important tonote that nano- sized and submicron-sized systems are thermodynamically unstable and éo not form spontaneously (Becher and Schick, 1987; Junginger, 1992; Capek, 2004) compated to microemulsions Which are thermodynamically stable and form spontaneously (Wennerstrom etal, 1897). Nano- and submicron-slzed emulsions have been used as carers for drugs and other bioactives, not only {or parenteral and ocular delivery, but also for topical and ddermatological-cosmetic applications (eg, vitamins and anti- aging agents) (Benita and Levy, 1993: Calvo etal, 1996: Fernandez fetal 2000; Nicolaos etal, 2003; Ortet al, 2002: Piemiet al. 1999; Sonneville-Aubrun and Simonet, 2004; Sznitowsa etal, 2001; Tamilvanan, 2004; Yilmaz and Borchert, 2005). Some specific advantages of nanoemulsions for dermatological andjar cosmetic application ar: (i) theirsmall droplet size thatenables penetration * Coneponding author 3 schol of Pam, University of Connecti, 6 Nott Eagle Rod, tr, CT. 05255, United Seer, eli 1850486370 fast 60D 4a 4008 ‘mal addres dDurgessuccened (Barger. usppetotore nore pear 201462004 (5785170 201 Elser BY. alight reserved, (© 2014 Elsevier BY. Al rights reserved, through the stratum corneum, enhancing drusiactive penetration; (i) their Large surface area that allows faster release of drugs) Actives ontothe skin; and (i) their transparent aspect, ity. and ‘relative physical stability (even in an absence of thickener agents) that provides pleasant aestheticaspect and skin feel (Tadros etal, 2004; Sonneville-Aubrun and Simonnet, 2004), [Nanoemulsions are an appropriate vehicle to deliver vitamin E and Its esters as well as other ipephilie vitamins onto the skin. Vitamin E acetate, a semi-synthetic acetate ester of naturally occurring a-tocopherol, is known as an endogenous cellulat antioxidant (Tamburic and Abamba, 1999). In terms of dermato- logical-cosmetic applications, vitamin E can protect the skin from LUV light damages, reducing the appearance of fine facial ines and wrinkles, and thus helping to delay the progression ofaging(Ropke etal, 2003; Sorg et al, 2001; Tamburic and Abarnba, 1999; Zhai etal, 2005), Vitamin E molecules have a large hydrophobic alkane ‘chain, with an ether group and a phenol group that can each participate in hydrogen bonding with water. However, the overall nature of the molecule is hydrophobic with poor water solubility (S1E"" ei at pH 6, 25°C) (log P=1207 +027) (Dubbs and ‘Gupta, 1988; SciFinder Scholar®), [Nano~ and submicron emulsion systems are useful as delivery vehicles, as long as they have adequate stability and releate the active ingredient in an appropriate manner. Accordingly, it is necessary to conduct pre-formulation and formulation character zation studies ofthe active and ofthe emulsion system during the Intended sheltite and under the release testing conditions, For example, during the development of in viro release testing “6 4 Moras Dire ad Dress inerairl oul of Phaocets 45 (204) 455-462 methods itis important to establish the following properties: () ‘model drug solubility in the release medium so that sink conditions can be calculated and maintained throughout the experimental period; (i) model druglactve partition coefficient between oil and water phases; i) theoretical and actual drugloading; (iv) free drug and micellar solubilization; and (v) encapsulation efficiency (Washington, 1990; penita and Levy, 1993). ‘The curent United States Pharmacopeia methodology for analysis of vitamin Eand its esters requires saponification, liquid- Tiguid extractions and solvent evaporation (US Pharmacopela 33, [NF 28, 2009). This procedure has inherent drawbacks such af interference from formulation excipients. analyte degradation, and emulsion formation. In addition, iis a relatively complex multi step process with many possible sources to error (Scalia et al 1905), Feasible HPLC/UV methods for the detection and quantifi- cation of vitamin Eand other lipophilic vitamins in plasma Alvarez and Mazancourt,2001; Gimeno etal, 2001; Hartmann etal, 2007; Julianto et al, 1998) and in hydrophilic gel-cream formulations (Guaratini et al, 2006) have been described in the literature. However, the usual methods to analyze drugs[actives in lipid ‘emullsion-based formulations include extraction of the analyte from the formulation using organic solvents (eg, hexane, chioro- form) followed by quantification using normal phase chromatog- raphy. These processes are time consuming and generally allow oor separation ofthe drug from the formulation excipients (Gupta etal,.2005; Scaliaet al, 1995) Accordingly a reliable and practical analytical method forthe detection and quantification of vitamin E and its esters in emulsion-based formulations, without interfer tence of other emulsion components (ie. ol phase and surfactants) fs warranted. ‘The aims of this work were to: 1) determine the critical physicochemical proprieties of vitamin F acetate in canola cil water nanoemulsions (i. vitamin E solubility, partition coefficient between emulsion phases loading, percentage of free vitamin E and encapsulation ellciency); and 2) develop a reliable and practical reverse phase liquid chromatographic analytical method (RP-HPLC) for the estimation of vitamin concentration in the ‘canola ol nanoemulsions. Macro-and micrescopie stability studies ‘ofnanoemulsions under stress conditions and accelerated stability tests were also performed. 2. Materials and methods 2. Materials Emulsions vere prepared using canola ll commercial grade) purchased from Bunge (St touls, MO)-The surfactants used were PEG-A0 hydrogenated castor oil (My=2589) (CemophorRl¥40") Kindly donated by BASF (lobam Park, N)) and sorbitan mono cleats (My =428.61)(Span80") purchased from Sigma-Aldrich (St touts a0), Double esta water was used as he water phase (Nanopure® Utrapure, Banstead, Dubuque, IA) The fllowing solvents: methanol, So-propanl,tetrahydrfurano (TH) were purchased ffom Fisher Selentfe (Pitsburg, PA). Phosphoric acid Solution (10% viv] was purchased from Labchem Ine. (Pitsburg, BA) and sodium acetate tlhydrate was purchased from Sig- ‘ma-hldrich (St Lous, MO}. Labrasol® was kindly donated by Gateffos SAS US (Paramus, N)). Microcon centrifugal Alter levies with Ulrace® YM-50 membranes (regenerated cellulose Oka MICO) were purchased ffom Milipore (Bilerca, MA} SpectalPor® DispoDialyer dialysis sacs (cellulose ester mem branes, MWCO 10kDa, 2m were purchased from Spectrum Labs (Rancho Dominguez, CA). Vitamin E acetate (tocopherol acetate, ‘96%) was purchased from Sigma-Aldrich (Lot 1OOT7AE) (St. Louis, MO), Chemicals were used as received without further purifiation. 22, Preparation of nanoemulsions Nancemulsions were prepared by the wask-out method previously developed (Moras etal, 2006) in which the water phase is continuously added into the oil phase (canola cil) Containing the surfactant blend and the model ative (vitamin E cette), Initially both surfactants (Span80® 242% and CremophorRi40" 2.58%) and vitamin E acetate (208 ww) were dissolved in the oil phase (.0% w/w) (anola iD, The water ad ei ‘Phases were separately preheated upto 761°. The water phase {G0.0% ww) was sowty added nto the oi phase containing the blend of surfactants and the model active. The emulsfestion temperature was set at 74°C. The components were mited sng mechanical stirring at 400 rpm (Yamato LR®,Lab-Stirer LR&00D, Santa Clara, CA) until the emlson temperature cooled to room temperature (25°C). ‘The surfactant ratio was chosen to achieve the required HLB for «anolacil(88), (previously determined, Morais etal, 2006). (1) was used to eieulate the amount of each surfactant needed to achieve this desired emulsion HLB (Becher and Schick, 1987): HUB = HIB, x 0.014 + HLBs x 0.015 ® where: HLB=emulsion hydrophilie-lipophilic balance, A= amount of hydrophilic surfactant in % (w/w), B=amount of lipophilic surfactant in % (wlw), A+B= 100%, 23, Emulsion characterization 2.31, Macro- and microscopic analyses ‘Macroscopic analysis (visual observation) of the vitamin E scetate nanoemulsions was performed in order to observe any ‘macroscopic instability such as creaming, phase separation or color changes. Vitamin E acetate nanoemulsion droplet size and size distribution were measured using a dynamic light scattering pafticle sie analyzer (Wicomp™ 380 submicron particle size analyzer, Santa Barbara, CA) equipped with a laser (wavelength {632.8nm) set atan angle of 90° and at 25°C. Nanoemulsions were diluted 500-fold either in distilled water or CremophorRH40® solution (258% wiw) before droplet size estimation. Nano- ‘emulsions were maintained either at 254:01°C or 32+01°C (Gsotemp Standard Lab Incubator, Fisher Scientific) throughout the ‘experiment and the macro-and microscopic analyses were carried ‘out using freshly prepared emulsions and at the following time point: 1,714 21,28, 42,60 2nd 98 days after preparation (for 25°C ‘ondition) and 1,7, 14,21, 35,53 and 91 days after preparation (for 52°C condition) as described above. The influence of the concentration of active (either 0.5 or 2.0%) was also evaluated. ‘The results (volume weight distribution) were reported as meaniSD (n~3). Student's -test with the level of significance st povalue < 0.05 was used. 232, Accelerated stabiley test ‘Accelerated stability tests were conducted as follo (i centrifugation test: immediacely following preparation, sa ples (100ml) were taken and submitted tothree diferent rotation speeds (1000, 2000 and 3500rpm, 170, 670 and 2000 xg, respectively) (Beckman Coulter Allegra X-15R centrifuge, Santa Clara, CA) one alter the other for 30minutes at each speed (@5=01°C); and (ii) thermal stress test: immediately after preparation, vials containing 25.0g of sample were submitted to 2 range of temperatures (from 40 up to 80:£0.1°, increasing by 5°C intervals) for 30min at each condition. The temperature was adjusted using a thermostatic water-bath (Microprocessor con- trolled 280 series, Thermo-Scientific), The macro-and microscopic stabilities of the vitamin E acetate nanoemulsions were observed immediately after emulsion preparation, 24h after preparation |. Maras Diane a) teratrl onl of Maracas 5 (2014) 455-469 7 nd following the end of the tests (see methods above). The results (volume weight distribution) were reported as mean + SD (n=6) and were evaluated using the Student's T-test with the level of, significance at p-value <0.05, 24, Development of the analytical method ‘The absorption spectrum was recorded between 210 and 400m (Cary $0 Bio UV-visible, VARIAN). UV scan testing were performed in order to verify the maximum absorption wavelength (for vitamin € acetate, canola ol (ol phase), CremophorRH40 ‘and Span80® using methanol, io-propanol and THF as solubilizing media. The objectives were to: () determine the strongest i ‘maximum which would then be used for the HPLC method, since different values are reported in the literature (Alencastre eta 2006; Dubbs and Gupta, 1998; Guaratin et al, 2008; Sealia et a 1895): and (i) verity possible interfering peaks (overlapping) of the oil phase constituents (which contains eg. a-tocoferol) (Ving and DeMan, 1989) and ofthe surfactants on the drug detection peak. Samples were prepared by dissolving known amounts of vitamin E acetate, canola oil, CremophorH40® and Span80° In methanol, iso-propanol and THE. The results were reported as mean SD (0-3), Imorder to prepare the mobile phase for the RP-HPLC method in development, parameters such as capacity factor. retention time, ‘as well as column efficiency were considered, For the choice of Solvents, the following physicochemical properties were evaluat- cc: dielectric constant, density, surface tension, eluent strength and viscosity Different ratios of methanol, iso-propanol, THF and, phosphoric acd solution (10% vjv) were investigated. The results ‘were reported as mean 45D (n=) 2.44, Statistical tests and validation procedures Linearity of the detector response was verified using increasing amounts of vitamin E acetate (with thee diferent inital standard, solutions), Calibration solutions consisting of a range of concen- trations of drug (078 to 100.00 ug/ml) were prepared. Peak areas land retention times were measured and a calibration curve plotted. Linear regression analysis was caried out. The selectivity, Of the developed RP-HPLC method for the determination of vitamin E acetate nanoemulsions was investigated t the retention, times of the analyte. The medium to be used in the in vitro release experiments and as well as nanoemulsions without vitamin E acetate (blank nanoemulsions) were submitted to chromatograph- ie analyses (Alencastre et a 2006). Analysis of blank formulation is essential for accurate qualitative and quantitative results. in order to confirm the selectivity of the method for vitamin E acetate, forced degradation studies were cartied out in Labrasot® acetate buffer (pH 5.5) at 32+01°C (Gupta et a, 2005). ‘The limit of detection (LOD) and the limit of quantification (L0Q) were determined ffom the standard deviation of the esponse using known concentrations of vitamin E acetate. in order to determine method efficiency, the percentage recovery was calculated Samples were prepared by spiking nanoemulsons with known amounts of vitamin E acetate in iso-propanol. The recovery percentage was determined by comparing the peak areas of the vitamin E acetate extracted ffom the blank nanoemulsions with peakareas obtained after direct injection of equivalent amounts of ‘analytes (Scalia et al, 1955). The data are presented as mean values SD (n=3)-The significance difference was evaluated using Student's t-test and one-way ANOVA (single factor) with the level of significance at p-value < 0.0, 242. Vitamin E acetate analysis Vitamin E acetate was analyzed using the RP-HPLC method developed. The optimal HPLC conditions determined were as follows: mobile phase iso-propanol: methanol: THF (47.5:476:48) With 1% of phosphoric acid solution (10% vjv); low rate= 0.7 ml] nmin; 4=284nm, injection volame=20 jt; running time: 10min, A ‘Zorbax® Rx C18 column (446mm x 15cm) was used. Vitamin E acetate was detected using a PerkinElmer 785 UV-vis detector, The ‘mobile phase was filtered using a O45yim Slter (Type HVLP, Millipore) and sonicated for 30 minutes 25. Formulation properties £25, Solubility of vitamin B acetate in the receptor medium ‘The solubility of vitamin E acetate in three different media was determined in triplicate by adding an excess amount of vitamin & acetate to 200ml of media. The samples were maintained at 532::01°Cina shaker water-bath (New Brunswick Edison, N)and fotated at 100 rpm until concurrent values were obtained, Filtered and analyzed, Vitamin E concentration was determined by HPLC (ee above). The following media were investigated: (1) acetate Dulfer(pH..5); (2) acetate buffer (pH 5.5): ethanol at(50:50 viv) and (3) Labrasol® acetate buffer (pH 5.5) at different concen- tuations (10, 20 and 5.0% w/v), The lineatty of the detector response was checked using increasing amounts of vitamin E acetate (with three diferent initial standard solutions) for each ‘media tested, Calibration solutions consisting of a range of concentrations of drug (0.78 to 100,00yg/ml) were prepared. Vitamin Eacetace was analyzed using the HPLC method developed as describe above. The results were feported as mezn-=SD (n=), 252. Partition coefficient ‘The patttion coefficient P of vitamin E acetate between the aqueous and oil phases was measured by homogenizingiequll- bratin the folowing hydrophiliclipophilic solutions: (1) acetate Duler pH 5.5/canola ol containing 2.0% (w/w)of vitamin E acetate, (2) acetate butter pH 55 containing 258% (w/w) of ‘GremophorRH40® canola il containing 2.0% of vitamin E acetate, anu (3) acetate buffer pH 5 5/canola oil containing 2.0% of viamin E acetate and 2.42% (w/w) of Span80®, The surfactant ratio reproduces the olw nanoemlsion HLB canola required HLB~83). ‘Samples were homogenized using magnetic stirring for24 hours at ‘oom temperature (25°C) to allow phase saturation. olljwater phase ratios of 1:1 and 1:18 (which reproduces the emulsion phase Tatio) were investigated. The dispersions were then equilibrated uring 24h prior to sample centrifugation (20min at 3500rpm, 2000 x z) and filtration (PVDF type filter (045 }). The concentra~ tion of vitamin E acetate in each phase was measuted by HPLC(see above). The results were reported as mean +SD (n=3). The log P ‘was calculated according to equation (2) (Rtschel and Kearns, 1999): age = tog (£2) @) ‘where: Co=final vitamin E acetate concentration inthe oil phase, Gyevitamin E acetate concentration in the aqueous phase. 253, Determination of encapsulation efficiency “The encapsulation efficiency (EE) of vitamin E acetate in the nanoemulsions was determined by ultrafiltration, Centrifugal filter devices (Microcon. Ultracel YM-50, 50kDa MWCO) were used in order to determine the amount of the model active inthe ‘external phase of the nanoemulsions (free drug). Samples were submitted to 500% (5500rpm) during 30 minutes (Beckman Coulter Allegra X-15R centrifuge). 4 yl was withdrawn from the pellet and diluted using iso-propanol. Subsequentiy, the samples ‘were filtered using a PVDE type filter (0.45 .) and injected inthe HPLC (see above) for the estimation of vitamin E acetate ‘concentration. = Mec ae andj argentina una Pharmacets 465 201) 45-469 “The amount of encapsulated vitamin E acetate was calculated by the difference between the total amount of vitamin Eacetate used 10 prepare the nanoemulsions and the amount of vitamin Eacetatethat remained in the aqueous phase solubilized in micelles (Ea (3). EE ~ (Totalamountof vitamin Eacetate free amount of vitamin Eacetate) eo 26, Total amount of vitamin acetate Uttrafitration was also performed in order to estimate the amount of vitamin E acetate released into the external aqueous phase as a function of time, to evaluate emulsion stability. The following time points were investigated: 1,7, 14.21, 28,60 and 98. Droplet size measurement of the supernatant was’ performed immediately after ultrafiltration to evaluate possible size increase after the centrifugal stress (see method above). The results were reported as mean SD (n=3) 2.61, Determination of drug lading “The amount of vitamin E acetate in the ofw nanoemulsions was monitored as a function of time and thermal sess in order to verify whether possible decrease in the active concentration correlated to chemical degradation. Samples were maintained at 25 and 32°C and analyzed atthe following time points: 1,7,14, 21, 28, 42, 60 and 98 days after preparation (for the 25°C condition) and 1,7, 14,21, 35, 83 and 9Y days after preparation (or the 32°C condition) 4 of the nanoemulsions were dissolved In 2ml of tither THE or lso-propanal inorder to determine the total content fof vitamin E acetate in the formulations. Samples were filtered ‘sing a PVDF type filter (0.45j}. Vitamin E concentration was {determined by HPLC (see above). The results were reported as mean 48D (n=3). 262. Determination of vitamin £ acetate concentration in the temuision extemal aqueous phase as a function of ime ‘Since drug tention tudlescan contribute totheunderstandingof the colloidal formulation microstructure (Washington, 1900), the

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