Formulation and Characterization of Microemulsion Made by the Combination of Carvedilol and Fluconazole

Microemulsion

Microemulsions are isotropic, thermodynamically stable, transparent (or translucent) systems of oil, water and surfactant, frequently in combination with a co-surfactant with a droplet size usually in the range of 10-100 nm. These homogeneous systems, which can be prepared over a wide range of surfactant concentration and oil to water ratio, are all fluids of low viscosity. Microemulsion as drug delivery tool show favorable properties like thermodynamic stability (long shelf-life), easy formation (zero interfacial tension and almost spontaneous formation), optical isotropy, ability to be sterilized by filtration, high surface area (high solubilization capacity) and very small droplet size. The small droplets also provide better adherence to membranes and transport drug molecules in a thermo-dynamically stable spontaneously formed mixtures of ‘oil’ and ‘water’, the term “oil” actually means any water insoluble (hydrophobic) organic liquid [53-56]. Though water has been predominantly used as polar phase for the formation of microemulsion, recent studies have also shown successful application of ethanol as polar phase for formulating hybrid fuel systems having aforementioned structural similarities with ‘microemulsions’. The higher GCV of ethanol than that of water may be another advantage of incorporating it into the system. Micro emulsification approach is successfully applied in fields like separation, chemical reactions, nanomaterial preparations, and drug delivery. The preceding decades have witnessed dramatic increase in microemulsion based applications. Micro emulsification today plays a pivotal role in thrust areas like fabricating nanoparticles, oil recovery, pollution control, food and pharmaceutical industries etc.

2 Experimental Work

2.1 List of materials.

Instruments List

2.2Table 2. Details of Instruments used in the research work

2.3 Method of estimation of fluconazole and carvedilol.

Determination of UV absorption maxima for fluconazole and carvedilol.

10 mg of Ibuprofen was first dissolved in ethanol (10 ml) and was then carefully diluted upto 100 ml with phosphate buffer, pH 6.4 diluted to obtain a stock solution of 100 µg/ml concentration. Then from the stock solution, 1 µg/ml and 2 µg/ml test solutions were prepared using above diluting solvent, i.e., Ethanol to phosphate buffer (pH 6.4) (1:9). The solutions were scanned in spectrum mode for absorbance between 200-600 nm using UV-Visible Spectrophotometer. 10 mg of Zaltoprofen was similarly dissolved in 10 ml of ethanol and diluted upto 100 ml with distilled phosphate buffer, pH6.4 (100 ppm) and then scanned between 200 nm and 400 nm using UV visible spectrophotometer.

2.4 Preparation of Calibration curve. UV Method.

From the stock solution of fluconazole and carvedilol 100 µg/ml, test solutions of concentration 2, 4, 6, 8, 10, 12, 14 µg/ml were prepared by suitable dilution with hydroalcoholic solvent (Ethanol: PB pH 6.4, 1: 9). The absorbance of each test solution was then taken at the absorption maxima (222 nm) against hydroalcoholic solvent as a blank for Ibuprofen and 228 nm for Zaltoprofen. The above procedure was performed in triplicate and average of the absorbance was taken into consideration (Yousry M, 2021).

2.5 HPLC Method.

Ibuprofen in solubility study samples as well as in brain and blood samples was quantified using the developed and reported reversed phase high performance liquid chromatography (RP-HPLC) method (Albert K, 2021 and Choudhary S, 2022). HPLC analysis was performed by using Waters 515 Series pumps combined with a detector i.e., Waters PDA 2998 series photo diode array (DAD). The column so used was Agilent C18 column (150 mm×4.6 mm, particle size 5-micron Agilent, USA). Analysis was done isocratic at 0.8 ml/min flow rate with mobile phase i.e., ACN: Buffer (0.025M Potassium dihydrogen ortho phosphate) pH 4.5 at 11:9 v/v. First the mobile phase was prepared by mixing the above said components and filtered through a 0.2 µm nylon membrane filter followed by degassing by sonication before use. The elution of drug was done at 230 nm. For Zaltoprofen, the experimental conditions were Acetonitrile and Phosphate buffer pH3 (60:40 v/v), as mobile phase at a flow rate of 1 ml/min and at 254 nm. HPLC column used was 5µm intensil, C18 column (4.6 x 250mm x 5µm).

2.6 Selection of Mobile phase and Preparation of Solutions.

Based on the literature review and solubility study, the mobile phase with their ratio was selected.

2.7 Preparation of Standard Stock Solution.

Accurately weighed Carvedilol as well as Fluconazole (10 mg) was dissolved in 100 ml of respective mobile phase taken in 100 ml of calibrated volumetric flask to prepare a solution of 100 μg/ml the same was further diluted into 100 times. An aliquot of 5 ml was pipette out from the stock solution and transferred to the calibrated volumetric flask of 25 ml and diluted to the mark in border to prepare 200 ng/ml using their respective mobile phase as diluting solvent.

2.8 Preparation of Working Standard Solutions.

For both the drugs, different aliquots of 1.0, 2.0, 3.0, 4.0 and 5.0 ml from the prepared stock solution (100 ng/ml) were separately transferred to a 10 ml of calibrated volumetric flask and dilute up to mark to generate the solutions of 20, 40, 60, 80 and 100 ng/ml concentrations with mobile phase. The test solutions were sonicated and further filtered through 0.22 μm filter i.e., nylon paper.

2.9 System Suitability Testing.

It can be defined as the tests to ensure the accuracy and precision of the developed system. The defined parameters in USP are used i.e., plate number (N), tailing factor (tF), resolution Time (RT) and the relative standard deviation (% RSD) of the peak height or the peak area for repetitive injections (n=5). At least two of these criteria are necessary to demonstrate system suitability for any methd

Table 3. Parameters for System Suitability

2.10 Preformulation study.

Identification of fluconazole and carvedilol.

Melting Point.

Melting point of both drugs was determined by capillary method through a melting point apparatus. A small amount of drug was inserted into the capillary tube (closed at one end) and the same was then placed into the melting point apparatus. Finally, the temperature at which drug starts melting, was taken as melting point and was then compared with the reference data (Adamo F, 2009).

2.11 FTIR spectrum of Ibuprofen and Zaltoropfen.

FTIR spectroscopy is a useful tool for the structure elucidation of drug using KBr disc method. The pure drug sample was triturated and dispersed with micronized IR grade KBr powder followed by application of 8-12 kpa pressure in the hydraulic KBr press to prepare the pellets. Pure KBr powder was used as blank for baseline correction. The pellets were transferred to sample compartment and were then scanned in the region of 400-4000 nm by FTIR spectrometer. Comparision of obtained FTIR spectrum of test sample was made with reference Ibuprofen as well as Zaltoprofen spectrum (Matkovic S, 2005).

2.12 Percentage Purity.

Percentage purity was done as per procedure given in IP 2010, the given limits (85% to 115%).

2.13 Spectral analysis (Absorption maxima, λmax determination).

50 μg/ml of Ibuprofen as well as Zaltoprofen solution was prepared in hydroalcoholic solvent. The absorbance was determined by scanning the sample at entire range of UV range (200-400 nm). The same was compared with the reference (Hapse S, 2011 and Diana M, 2010).

2.14 Solubility profile of fluconazole and carvedilol.

5 ml of different solvents (distilled water, phosphate buffer pH 6.4, acetonitrile, ethanol and acetone) were taken separately in the test tubes and excess amount of drug was added to saturate the system. The dispersion was shaken in thermostatic orbital shaker for 48 hrs at 37 ± 2˚C (80 rpm). All samples were then centrifuged at 8000 rpm for 15 min. To obtain only solubilised Ibuprofen as well as Zaltoprofen, the supernatant was filtered through 0.45μm filters and the filtrate was finally analyzed using UV-VIS spectrophotometer at appropriate wavelength (Diana M, 2010 and Yan et al, 2009). This test was performed three times and results were taken as mean ± SD.

2.15 Screening of components.

The screening criterion to select mucoadhesive microemulsion compositions are as per the literature (Jing C, 2006 and Huabing C, 2006).

• All formulation compositions should be of GRAS (Generally recognized as safe) category for intranasal administration.
• Drug solubility for oil phase, surfactant and co-surfactant (Jing C, 2006).
• Non-irritating property of surfactant and co-surfactant.
• HLB value of surfactant i.e., 12-16 for the development of O/W microemulsion.
• Drug excipients compatibility property (Chen et al, 2006).

Basing on the biocompatibility profile of oils for nasal mucosa from the literatures, drug solubility was determined. Excess amount of drug (100mg) in 5 ml of selected oils (Isopropyl Myristate, Oleic acid, Maisine 35-1, Capryol 90, Labrafil WL 2609 BS, Capmul MCM EP, Capmul MCM, Labrafil M 1944CS, Labrafac PG and Labrafil M 2125CS) was taken in stopper vials (10 ml) and were then mixed by vortex mixer. The vials containing mixture were then kept at 37 ± 2.0 C and 80 rpm in orbital shaker at isothermal condition for 48 hr to reach equilibrium. The equilibrated samples were the centrifuged at 5000 rpm for 15 min. The solubility profile of drug in oil was finally determined from the supernatant by using UV-VIS spectrophotometer at its respective absorption maxima (Serajuddin A, 2021). Various surfactants with HLB from 12 to 16 (Tween-80, Tween-60, Labrafac CC, Acconon CC, Cremophor EL and Cremophor RH 40) were screened for drug solubility in the same procedure as described for oils (Jing et al, 2006 and Zhang P, 2008).

2.16 Drug-Excipient compatibility study.

Drug-Excipient compatibility study was performed to ensure that the formulation has relatively more biocompatibility and physical stability of the formulation. Adequate quantity of drug to individual formulation components mixed separately and was kept undisturbed for 7 days. At day 3 (D3) and day 7 (D7), the samples were checked for precipitation, phase separation and color change in order to determine their physical compatibility (Pani N, 2011). Chemical compatibility is basically regarded as the chemical stability of the drug in all formulation components. Surfactant, oil and cosurfactant were considered for further development only if physically and chemically compatibility with drug was observed. Adequate quantity of Ibuprofen and all formulation components mixed and was kept at room temperature and at 50 C for 7 days FT-IR spectra of the mixture at day 1 (D1) and day 7 (D7) were analysed for compatibility since FT-IR spectrum gives the identification of specific functional groups and so that from this any chemical incompatibility if occurred can be easily identified by change in the peak of functional group (Marini et al, 2003).

2.2.1 Preparation of fluconazole and carvedilol microemulsion formulations.

Water titration technique was used for developing mucoadhesive microemulsion with the selected oil phase, surfactant-cosurfactant mixture and aqueous mucoadhesive polymeric phase (Patel M, 2013 and Acharya S, 2013). Required quantity of fluconazole and carvedilol was added to adequate amount of oil separately in a cleaned and dry beaker having a small magnetic bead and was mixed completely using magnetic stirrer. Then, to the mixture, surfactant and co-surfactant at definite ratio (Smix from the pseudo-ternary phase diagram) was added and was mixed at same rpm. The above mixture was finally titrated using aqueous solution of mucoadhesive polymer under same experimental condition on a magnetic stirrer (Omar S, 2012).

Table 4. Levels of Process variables in 32 factorial design

2.2.2 Transparency (% Transmittance and Refractive index).

Both developed MMEIs and MMEZ were diluted 50 and 100 times with distilled water. Optical transparency of these formulations was quantified at 560 nm by UV-VIS spectrophotometer against water. Results were taken in triplicate and the average was taken in to consideration (Chandra A, 2009). Abbe’s refractometer was used to determine the refractive index of the formulation. Results were taken in triplicate and the average was taken into consideration (Mandal S, 2011).

2.2.3 Globule size and Zeta potential analysis.

Both developed MMEIs and MMEZs were diluted 50 times and 100 times with distilled water by gentle agitation for 5 min spin vibrtor. In addition, globule size distribution (PdI) were determined using dynamic light scattering technique while zeta potential or surface charges of MMEIs was determined by Malvern zetasizer (NANO ZS). Results were taken in triplicate and the average was taken in to consideration (Joakim B, 2010 and Patel R, 2012).

2.2.4 Viscosity analysis.

2.2.5 In-vitro drug release study.

In-vitro drug release     study    of microemulsion formulations [MMEI(C) and MMEI(L) as well as MMEZ] were carried out in Franz diffusion cell having volume of 30 ml and an effective permeation area of 7.06 cm2 containing 30 ml of dissolution media i.e., ethanolic phosphate buffer saline (PBS), pH 6.4, as used in the UV determination (Pathak R, 2014 and Sharma G, 2009). Temperature was maintained at 37 ± 0.5 0C and rpm was set 50. Dialysis membrane of cut off weight 10,000 D was soaked PBS (pH 6.4) overnight prior to experiment.

2.2.6 Release kinetics.

In order to study the release kinetics of fluconazole and carvedilol from developed MMEs, the release data so obtained were fitted to the following equations as per the literature (Starychova et al, 2014 and Carvalho et al, 2010) and reference data is given in Table-2.2.7 Zero kinetic equation: Qt = k0t, Where, Qt- % drug released at time t and k0 - Release rate constant.

First kinetic equation: ln (100-Qt) = ln 100 – k1t, Where, k1- Release rate constant.

Higuchi’s kinetic: Qt = kHt1/2, Where, kH - Higuchi release rate constant.

Hixson-Crowell kinetic: (100-Qt) 1/3 = 1001/3 – kHCt, kHC – Hixson-Crowell rate constant.  Korsmeyer Peppa’s model: Qt/Q∞= kKPtn, Where, Qt/Q∞ - Fraction of drug released at time t,   kKP a constant and n, the release exponent indicating the drug release mechanism.

Table-5. Release kinetics (Diffusional exponent v/s type of release)

2.2.9 Stability studies.

Stability of fluconazole and carvedilol mucoadhesive microemulsion.

Samples of Ibuprofen mucoadhesive microemulsion (MMEI) formulations i.e., MMEIs and MMEZ were put into ampoules and then kept in stability chambers at different experimental conditions i.e., Cool temperature (4 to 8 0C), room temperature (25 0C) and elevated temperature (40 ± 2 0C) with different relative humidity for 6 months. One sample was withdrawn at 3 and 6 months to evaluate their physical and chemical stabilities. Physical changes like color change and phase separation were checked through visual inspection for the physical stability of the developed MMEs (Mandal S, 2010). Chemical stability of MMEIs and MMEZ was also determined by determining the fluconazole and carvedilol content using UV-VIS spectrophotometer at 222 nm and 228 nm respectively. Globule size and size distribution using Zetasizer were also measured and retension time (min) in order to evaluate the chemical stability profile of the drug and dosage form (Makhmalzadeh B, 2012). Developed MMEZ was kept in the stability chambers in same way that of MMEIs and same parameters were also evaluated.

3 Preformulation Studies

Pre-formulation studies are preliminary studies to understand physicochemical behavior of a new drug and possible hurdles in dosage form development. It generates supportive data for necessary modifications to design, develop and evaluate formulation.

3.1 Identity and Confirmation of Pure Drug Preliminary evaluation

The fluconazolesample was evaluated visually for appearance, color, odour and taste. Physical evaluation like color, odor, solubility and determination of melting point performed to confirm identity. Melting point determination of the pure drug sample was done, as it is a first indication of purity of the sample. It was determined by melting point determination apparatus.

Drug:               fluconazole

Colour:           Colorless

Odour:              Odorless

Solubility:        Ethyl acetate

Melting point: 2200C

3.2 Chromatographic evaluation

Thin layer chromatography (TLC)

Thin layer chromatographic evaluation was done to add more confirmation to identity. This chromatography is useful to separate compounds and their retention factor in specific stationary and mobile phase on comparison with authentic literature helps in identification and confirmation.

3.3 High Performace Liquid Chromatography (HPLC)

Chromatography was carried on a HypersilODS C18 column using a mixture of methanol and water (80:20 v/v) as mobile phase at a flow rate of 1.0 mL/min with detection at 280 nm. The retention times were 4.02 min. (Figure 4.2) The results complies with previous literature.

3.4.1 UVmax determination

In order to ascertain the wavelength of maximum absorption (λ max) of the drug, solution of the drug (10µg/ml) in Ethanol was scanned using spectrophotometer within the wavelength region of 400-200 nm against Ethanol as blank. The absorption curve showed characteristic absorption maxima at λ max 280 m for fluconazole. (Figure 4.3) All these preliminary evaluation studies confirm identity and purity of selected active pharmaceutical drug.  Spectroscopic and chromatographic method helps in estimation of drug content, drug release, drug entrapment efficiency and drug loading.

UV Spectrum of fluconazole in ethanol at 280 nm

3.4.2 Analytical Method for the Estimation of fluconazole in Ethanol

The absorbance of each concentration of 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 µg/ml was measured at 280 nm. Results are shown in a graph of absorbance versus concentration. It shows straight line meaning the calibration curve obeys Beers law. After scanning 50µg/ml solutions, maximum absorbance obtained at 280 nm and considered as λmax. From the standard curve, (Figure 4.4.) it was observed that the drug obeys Beer’s law in concentration range of 10-100 µg/ml in ethanol. The absorbance of each concentration 2, 4, 06, 08, 10, 12, 14, 16, 18 and 20 µg/ml was measured at 280 nm. Results are shown in Table 4.1. A graph of absorbance versus Concentration was plotted and it is shown in Figure 4.5. It shows straight-line meaning the calibration curve obeys Beers law.

Table 4.1: Standard calibration curve of fluconazole in ethanol at 280 nm *(n=3)

Standard calibration curve of fluconazole in ethanol

Standard calibration curve of fluconazole in phosphate buffer (pH- 6) at 280 nm.

3.5 Analytical Method for the Estimation of fluconazole in Phosphate Buffer (pH 6)

The absorbance of each concentration of 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 µg/ml was measured at 280 nm. Results are shown in a graph of absorbance versus concentration. It shows straight line meaning the calibration curve obeys Beers law. After scanning 50µg/ml solutions, maximum absorbance obtained at 280 nm and considered as λmax .From the standard curve, (Table 4.2.) it was observed that the drug obeys Beer’s law in concentration range of 10-100 µg/ml in. phosphate buffer (pH 6). Drug shown good linearity (Figure 4.4) with regression of coefficient (r2=0.9979) and equation for this line obtained was found to be (y =0.0016x - 0.0015) which is used for the calculation of amount of drug and diffusion study.

Standard calibration curve of fluconazole in phosphate buffer (pH 6) at 280 nm. *(n=3)

3.6 Solubility Study of Drug in Oil

To select the best oil for preparation of microemulsion formulation, saturated solubility studies were carried out in different oils, i.e. soyabean oil, castor oil, olive oil, labrafill 1944 and oleic acid etc. Excess amount of drug i.e. fluconazole added to the 200mg each oil in glass vial. Then mixture solubilizedby sonicator for 30 min. Further mixture containing vials were kept in orbital shaker for 72 hr. to from homogenous mixture. This was done by preparing saturated solutions of the drug in these oils and analyzing their drug content spectrophotometrically. [Kyatanwar AU 2010]

List of oils used for the solubility study and Solubility of fluconazole in various oils

Among all screened oils, the highest solubilisation capacity was exhibited by oleic acid (30.72±4.7430) followed by labrafil 1944 (16.7039±1.1145mg/mL) and castor oil (12.7105±1.28mg/ml). Therefore, oleic acid was selected for further investigation. Results of solubility are summarized in

To select correct oil to formulate microemulsion, determination of solubility of the drug in different oil phases like synthetic or natural is generally practiced method. O/W microemulsions solubilizes lipophilic drugs whereas w/o systems solubilizes hydrophilic drugs. For poorly soluble drugs, drug loading is a very critical design factor which depends on the drug solubility in oils.

Being non-ionic and hydrophilic in nature, Tween-80 is most suitable in formation of O/W microemulsion with less toxicity to skin. Non-ionic surfactants are also less prone to be affected by change in PH. Propylene glycol (PG) being lipophilic shows its critical role as co-surfactant compared to PEG and Transcutol-P in formation of microemulsion. Mixture of both hydrophilic surfactant and lipophilic co-surfactant can be good choice in formulating fluconazole drug microemulsion devoid of surfactant toxicity with more than 700 times more (30.72±4.7430 mg/ml) as compared to water solubility of fluconazole (0.0403 mg/ml).

3.8 Surfactant and Co-Surfactant Screening

The final selection after solubility analysis was done on the basis of HLB value of co-surfactant. Among all surfactant screened, the highest solubilisation capacity was exhibited by Tween 80(27.8289 mg/mL) followed by Cremophor RH 40 (26.1842 mg/ml)and Labrasol (23.8815 mg/mL). Tween 80 was therefore selected for further investigation, while final selection would rely on emulsification properties with co surfactant mixtures. For solubility studies surfactants and co surfactants were chosen from the GRAS (generally regarded as safe) category. Nonionic surfactants are reported to be less toxic than ionic surfactants. From the results of screening studies, it was observed that, Co Surfactant like Transcutol-P, PEG 400 found to have very good solubilising capacity compared to Propylene Glycol. (Figure 4.7) But as the solubility criteria is not key parameter for selection Propylene Glycol have HLB value in range of 4- 6and also having a permeability enhancer, selected co-surfactant also show good emulsification with selected oil and Tween 80. fluconazole was more compatible for emulsification ability with Tween 80 and Propylene glycol, hence Propylene glycolused as co-surfactants.

Table 4.4: Solubility of fluconazole in Surfactants and co surfactant *(n=3)

3.9 Drug-Polymer Interaction

The FTIR spectrum of fluconazole was shown in Figure 4.8. The FT-IR spectrum of fluconazole pure drug was found to be similar to the standard spectrum of fluconazole. Further drug-excipients (Figure 4.9) compatibility study was investigated by FTIR spectroscopy. Pure fluconazole shows major peak at IR spectra revealed no considerable change when compared that of NME (Figure 4.10) formulation proves that there is no interaction between drug and excipients. Here TLC and FTIR studies are done for individual active drugs and final optimized formulation. The spectrum of fluconazole showed the following functional groups at their frequencies:

Figure 4.8: FTIR spectrum of microemulsion without fluconazole

4 Pharmaceutical Evaluation of Formulation

Appearance

If a single surfactant film is desired, the lipophilic chains of the surfactant should be sufficiently short, or contain fluidizing groups (e.g. Unsaturated bonds). Short to medium chain length alcohols (C3- C8) are commonly added as cosurfactants which further reduce the interfacial tension and increase the fluidity of the interface. This gives uniform, clear, transparent microemulsion. Microemulsions were checked for transparency and turbidity by measuring absorbance at 600 nm. Microemulsions remained clear on dilution but appeared as transparent yellow colored solution due to presence of oils and surfactants. (Figure 4.34) All the formulation found clear and devoid of any sign of precipitation. [Zhao L et al., 2013]

4.1 Optical clarity (Percentage transmittance)

In this method, percentage transmittance of each formulation was measured at 600 nm using UV-spectrophotometer against distilled water as blank. Percentage transmittance indicates the homogenous nature and clarity of formulation.

4.2 All formulation showing transmittance in the range 98-100% (Table 4.24) and were found to be optically clear and transparent. High transparency is due to the droplet are much smaller than the optical wavelength of visible spectrum. [Celebi N et al., 2012]

Table 4.24: Percentage Transmittance and drug content of microemulsions

4.3 Type of emulsion (Dilutability and dye solubility test)

2-3 drops of water-soluble dye (methylene blue) was added  to the  microemulsion formulation and after 5 minute visual observation was done. Water-soluble dye such as methylene blue or amaranth is when added to emulsion and if drop is observed under microscope, background looks blue/red and globule appears colorless shows water in oil emulsion Generally external phase either the oil or the aqueous phase in the microemulsion preparation is used to dilute microemulsion. Hence, in case of o/w system the microemulsion can be diluted with the aqueous phase while with w/o microemulsion the system is diluted with the oil used. The Microemulsion formed were diluted in 1:10, and 1:100, ratios with double distilled water to check if the system shows any signs of separation. No trial batches showed any signs of separation. Stain test showed water in oil micro emulsion.

4.4 pH measurement

The pH of the formulation not only affects the stability of the emulsions but also alters the solubility and bioavailability of the drug through microemulsion at the site of permeation The pH value of ME was determined using digital pH meter (Equip-Tronics, EQ-610), standardized using pH 4 and 7 buffers before use. The pH of all the ME ranged between 5.5 and 6.5, (Table 4.25) approximating the normal pH ranges of nasal fluids, which is one of the formulation considerations that may help reducing the irritation produced upon instillation. pH of NME-2 was found 6.1± 0.05 The rheological properties of the microemulsion are evaluated by Brookfield viscometer. The surface tension of microemulsion was measured at 25ºC with a Torsion balance.It was observed that the viscosity of the ME formulations generally was very low. (Table 4.26) This was expected, because one of the characteristics of ME formulations is of a lower viscosity. Microemulsion NME- 2 shows low viscosity value of 110± 2.51cp respectively, Low viscosity of the formulation indicates that formulation is o/w type and having Newtonian flow ensure easy handling, packing, and hassle-free nasal administration of formulations. The surface tension data implies water-in-oil microemulsions because surface tension amounts of MEs are nearby to oil phase surface tension. [Gui SY et al., 2008]

4.5 Refractive index

The refractive index of the system was measured by a simple Abbe’s refractometer. The refractive index of the systems was found to be in range 1.40 ± 0.53 to 1.41 ± 0.7637. It reflect the microemulsion appear nearly transparent in the visible spectrum and exhibit very little scattering having a low refractive index. [Venkatesh G et al., 2010]

4.6 Droplet size distribution and zeta potential Determination

Measuring particle size distributions and understanding how they affect your products and processes can be critical to the success of many manufacturing businesses. Zeta potential is a measure of the magnitude of the electrostatic or charge repulsion/attraction between particles, and is one of the fundamental parameters known to affect stability. It has been suggested that ZP may serve as a partial indicator for the physical stability of the emulsion being formed.

4.7 High absolute ZP values (±30 mV) should preferably be achieved in most of the emulsions prepared in order to ensure the creation of a high-energy barrier against coalescence of the dispersed droplets. [Gui SY et al., 2008] Particle size, PDI and zeta potential are the important characteristics of the microemulsion. Microemulsion generally has a low particle size (< 200 nm) as compare to the emulsions. PDI of the particle should be less than 0.5 which indicate the prepared partials are mono disperse. [Zhou XT et al., 2008] Particle size analysis of six preparations showed that the size range lied between 149.8 to 193.9. PDI of N-1 to N-4 was found below 0.5 but N-4 and N-5 showed PDI more than 0.5. Zeta potential values found that formulation N-1 to N-6 were within the ideal range of -10 to -30 mV.

4.8 Conductivity measurement

The electrical conductivity of ME was measured with a conductivity meter (Equip-Tronics, EQ – 664, Mumbai, India) equipped with an inbuilt magnetic stirrer. This was done by using conductivity cell. NME-2 shows (Table 4.27) the conductivity measurements value 178 (ms/cm) indicate the water in a continuous phase and ME to be of oil-in-water type. Electrical conductivity is structure sensitive property, which is affected by the composition of a system along the dilution. To understand the phase behavior of ternary phase systems phase either separation or inversion according to change in composition or dilution, electrical conductivity measurement is key parameter. Low content of water in microemulsion shows low conductivity means o/w microemulsions possess high conductivity.

Results of conductivity measurement

Table 4.28: Results of Particle size, zeta potential

Figure 4.35: Partical Size and Pdi of Batch N 1

4.9 Entrapment efficiency and drug loading

Entrapment efficiency of drug depends on the method of preparation, HLB values, and chemical structure of the surfactant. The length of the alkyl chain of surfactants also governs the entrapment efficiency. Surfactants of longer saturated alkyl chains shows higher entrapment efficiency. Hence, use of Tween 80 expects same entrapment efficiency in this research work. The entrapment efficiency of fluconazole loaded microemulsion was determined by a centrifugation method. Drug loading is different from entrapment efficiency. Ratio of weight of drug entraped into carrier system to the total drug added while drug loading is the ratio of drug to the weight of total carrier system. Drug loading is physical entrapment. Particle size, excipient concentrations are important factors in determination of drug loading. Results are summarized in Table 4.29. The results of assay revealed suitability of the system for high entrapment of drug in the internal phase.

Percentage entrapment efficiency and drug loading

Drug content estimation

Microemulsions are getting popular due to alternative delivery system which is stable; spontaneously formation and improved maximum solubilisation of lipophilic drugs due to smaller particle size. Drug content estimation is one of the important parameter to know efficiency of developed delivery system which can be comparable to drug release data. Amount of loaded active drug should be in good limit to obtain expected therapeutic efficacy. Microemulsion equivalent to 10 mg of fluconazole was dissolve in suitable quantity of ethanol (100 ml). The samples were mixed thoroughly to dissolve the drug in ethanol and analyzed using Shimadzu 1800A UV visible spectrophotometer at 280 nm. Drug content was found to in range of 97.35% - 99.81 % during estimation which complies dosage form design expectation. This result also indicated that there was uniform distribution of the drug throughout the batches. (Table 4.30) Optimized batch N2 showed 99.24% of drug content.

Table 4.30: Results of drug content