Chapter Four — Methodology.
Preface
THIS WILL BE PUBLISHED PER CHAPTERS: PART 4/8.
During my final year of my bachelor of pharmacy, I had the joy of doing a project in pharmaceutics on topical corticosteroids. My colleague and I achieved a distinction for this project but we do admit that we can see some mistakes in our project. This project took us 6 months to accomplish and it mainly taught us about research, reading the literature and to attempt at making a pharmaceutics project. This was more of an introduction to a masters degree path and also taught us to work on our own and be supervised with an allocated supervisor. All in all, we learned a great deal and we were thankful for it.
Authors: Andréas L.P. Astier, Priyanka Naidoo
Supervisors: Professor R. Walker, Dr S.M. Khamanga
Location: Pharmaceutics Department, Rhodes University, Grahamstown, 6140, South Africa.
CHAPTER FOUR
Methodology
4.1. Introduction
In analytical chemistry, the accurate quantitative measurement of the composition of samples, such as in spectroscopy, would require calibrations using the standard samples of known composition (60). A calibration curve is an analytical method to determine the concentration of an unknown sample solution (60). This is done with known solution samples and standards dissolved in a suitable solvent (60). In the calibration curve method, a series of standard solutions are prepared and measured, usually with up to 5 points. A line would be created to fit the data and the resulting equation is used to convert readings of the unknown samples into concentration (60). The equation and the R2 value are calculated with Microsoft Excel 2016. An advantage of this method is that the random errors in preparing and reading the standard solutions are averaged over several standards (60). As such, the non-linear in the calibration curve can be detected and avoided, keeping the relationship true (60). The calibration curve can then be used in various analyses of various pharmaceutical formulations including semi-solid preparations such as creams, ointments and gels (61). The optimum consistency of such a formulation helps ensure that a suitable dose is applied or delivered to the target site (61). This is particularly important with formulations of potent drugs such as CBP (4, 16) which would be delivered as aqueous cream to the integumentary system. A reduced dose would not deliver the therapeutic effect, and an excessive dose may lead to undesirable and toxic side effects (61). Hence, the release of the API is an important aspect of the treatment of diseases and patient wellness.
Dissolution curve is used in analytical chemistry with the use of a spectrophotometer and is generally accompanied by its relevant calibration curve (60, 62). Ultimately, the dissolution curve demonstrates the bioequivalence and the dissolution profile curve of an API and its formulation which can be compared, if available, to the FDA, WHO, and relevant Pharmacopoeia (62). Spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength (63). In spectrophotometry, the use of visible light, near-ultraviolet, and near-infrared are utilized (63, 64). In the experiment, the UV mini 1240 spectrophotometer uses a 20 W halogen lamp as a light source (64). Spectrophotometry uses photometers that can measure a light beam's intensity as a function of its colour known as spectrophotometers (63). Important features of spectrophotometers are spectral bandwidth, the percentage of sample-transmission, the logarithmic range of sample- absorption, and the percentage of reflectance measurement (63). A spectrophotometer is used for the measurement of transmittance or reflectance of solutions, transparent or opaque solids, such as polished glass (63). This method was used to measure the amount of CBP released in the USP 2 apparatus 1000 millilitre vessels, and as such, with the aid of the calibration curve, a dissolution curve was obtained.
The efficacy of a topical therapy depends on the patient spreading the drug formulation in an even layer to administer a standard dose (59). Spreadability is, therefore, an important characteristic of these formulations and is responsible for correct dosage transfer to the target site which is highly dependent on the spreadability of the formulation, ease of application on the substrate, and the consumer preference (59). Semi-solid preparation common property is the ability to cling to the application surface for a reasonable period of time before they are washed off or worn off (59). A semi-solid preparation that can be spread easily over the wanted area and not over/under spread, is desired by patients which increases patient’s satisfaction of the semi-solid preparation. If over time the viscosity and spread changes from its original spread characteristics, it will influence the patient’s perception of the semi-solid preparation which may be negative. A change in viscosity indicates a change in the rheology properties of the formulation.
Chapter 3, Section 3.4., describes further the spreadability and visual and feel of the textural profile.
4.2. Calibration curve
A standard stock solution was made up of 0,1000 g / 1 L using a 1-litre volumetric flask on the day of the dissolution. 0,1000 g of powdered CBP, using the mortar and pestle, was weighed on the analytical scale up to four decimal places. Distilled water was added up to the 1000 ml mark. The solution was placed in a sonicator to ensure rapid dissolving of the CBP. A concentration of 100 μg/mL stock solution was prepared. Once the stock solution was ready 5 dilutions were prepared.
First dilution: 10 ml of stock solution was placed into 100 ml volumetric flask using a 10 mL pipette and distilled water was added up to the 100 ml mark.
Second dilution: 20 ml of stock solution was placed into 100 ml volumetric flask using a 10 mL pipette and distilled water was added up to the 100 ml mark.
Third dilution: 40 ml of stock solution was placed into 100 ml volumetric flask using a 10 mL pipette and distilled water was added up to the 100 ml mark.
Fourth dilution: 60 ml of stock solution was placed into 100 ml volumetric flask using a 50 mL measuring cylinder and a 10 mL pipette and distilled water was added up to the 100 ml mark. Fifth dilution: 80 ml of stock solution was placed into 100 ml volumetric flask using a 50 mL measuring cylinder and a 10 mL pipette and distilled water was added up to the 100 ml mark. Sixth dilution: Enough volume was taken from the stock solution to fill the quartz cuvette, no dilution was required.
All volumetric flasks were shaken until the solution was homogeneously dispersed. The first calibration curve included a concentration of 4 μg/mL, which was found to be too small and discontinued. All calibration curves contain their respective tables of dilution points and absorbance/concentration data.
Each dilution was placed into a clean quartz cuvette using a pipette and the absorbance of each of the diluted concentrations was measured using the UV mini 1240 spectrophotometer at 232 nm wavelength and recorded. A calibration curve was created and plotted graphically using the absorbance and concentration (mcg/mL) values. The linear line and regression line was calculated by using the Microsoft Excel 2016 program.
4.3. Dissolution curve
Dissolution tests are performed on the formulations to test the rate of release of CBP in all four formulations. The release of CBP from the aqueous cream was measured using an in-vitro diffusion cell apparatus. USP apparatus 2 consists of a paddle and vessel assembly and is used to conduct the dissolution of these formulations (23). The Erweka®GmbH type DT 620 was used as a USP apparatus 2.
The paddle assembly is made of inert plastic and is designed to create the least amount of interaction with any APIs, excipients and dissolution fluids (23). The cylindrical glass vessel contains a hemispherical bottom and a maximum volume of 1000 millilitres. 750 mL of medium solution was chosen according to the US Pharmacopoeia standards for the experiment. The temperature of this vessel is maintained at 37 ± 0.5oC which maintained optimum conditions during the test. The shaft is positioned so that the axis was not more than 2 mm from the vertical axis of the vessel at any point and should rotate smoothly without significant disturbances that could affect the results.
The jar, with the membrane and lid, was weighed on an analytical scale, and the weight was recorded. The formulation was then packed into the glass jar such that, air pockets were not formed inside the jar. Using tweezers, the membrane was soaked in distilled water and spread evenly over the formulation making sure not to form any wrinkles or air pockets. The lid was then screwed on, and the jar was measured and the weight was recorded. The diameter of the exposed membrane was measured for all four formulations. 750mLs of distilled water, maintained at 37 degrees Celsius, was placed in the vessel. The formulation jars were immersed into each of the vessels, with the jars resting on the base of the receptor vessels, with the membrane facing upwards. The agitating paddles were lowered to 1-2cm below the solvent surface. The paddle drive mechanism was engaged to agitate at a rate of 50 rates per minutes (rpm) to maintain near-sink conditions at the membrane surface.
At each sampling time interval, withdraw a specimen of 5mL aliquots from a zone midway between the surface of the medium and the top of the paddle, not less than 1 cm from the vessel wall. A 10 mL syringe for each different formulation was used at 10-minute intervals over 180 minutes and each aliquot was placed into a clean quartz cuvette. This was assayed by the UV mini 1240 spectrophotometer at 232 nm wavelength. The absorbance readings were recorded and the aliquots were returned to the bulk solution in the receptor beaker to maintain the volume of 750mL, hence not altering the concentration from removing volume.
Dissolution tests are employed to establish drug API release characteristics of semi-solid formulations (23). The rationale for conducting these tests is that for a product to be therapeutically effective, the drug must be released from the product (65). A dissolution test may be considered as a critical step for the development and assessment of the quality of products linking to their safety and efficacy attributes (65). An understanding between the interaction of hydrophilic like molecule and lipophilic like molecule is essential in the drug release from the semi-solid formulation into its environment (65).
4.4. Spreadability
The spreadability was measured using two glass plates of equal size with weights placed on top. 2 grams of the formulations were weighed by using the top loading balance and placed in the centre of the glass plate. The second plate was placed on top and two 100 gram weights are placed on top of the plate. This was done for exactly 5 minutes. The distance was measured with a 30 cm ruler, and an average and standard deviation were calculated. Each formulation spreadability was measured on Day 0, 7, 14 and 28 and a clustered column chart was made for representations of the data.
4.5. Textural profile
The four formulations were tested subjectively for the emolliency, viscosity, aesthetic appeal in terms of colour and smell; and texture such as smoothness and grittiness. These were observed, commented upon and noted out of 10. 0 indicated the lowest and 10 indicated the highest, such as in terms of viscosity 0 would indicate water-like viscosity and 10 would indicate a solid viscosity.
4.6. Degradation studies
4.6.1. Calculating the plateau
Each point considered on the plateaus of each formulation on Day 0, 7, 14, 28 and 60 were averaged and a standard deviation was calculated. These can be seen in Chapter 5, section 5.5., Table 5.10 - 14. Some plateaus were very small with only three points and others had up to seven points. The decision of taking in the points of what was considered to be on a plateau was discussed as a team and chosen. Hence, a more statistical or mathematical approach should be endorsed and is necessary to confirm if the points are significantly part of the plateau. This should be practised in future and similar experiments.
4.6.2. Plotting the plateau
The average, with the standard deviation, were plotted with its allocated time in days, seem in Figure 5.28. A non-linear line was produced to create a relationship between the average plateaus of percentage release in API per cm2 with time in days. As such, an exponential curve equation was calculated with the allocated R2 values, this is seen in Table 5.15. The exponential curve and the R2 values were calculated by using the Microsoft Excel 2016 program. The exponential curve equation was transformed into a natural log equation as seen in Table 5.15.
The natural log equation was plotted on a semi-graph paper with the natural log of average percentage release of API per cm2 against time. This produced a straight line as seen in Figure 5.29. The straight line produced on the semi-log paper can now help us predict the average percentage release of API per cm2. These were compared from theoretical and actual with the percentage difference from theoretical to actual (%), as seen in Table 5.16.
4.7. Accumulation studies
Based on the average percentage release of API per cm2 that was found in the experiment, the amount of API was calculated as to compare to a 100% average percentage release of API per cm2 found in the literature (4, 16, 20, 28, 29). Hence, the actual amount needed to use, from the formulations made, was calculated such that there is a correct and effective therapeutic effect. This can be seen in Chapter 5, section 5.6., Table 5.21. Further explanation is made before Table 5.21.
The actual amount needed for the correct and effective therapeutic effect of each formulation was plotted on a graph versus time in days as seen in Figure 5.30. The area under the curve was calculated manually and the data was plotted after each use, this can be seen in Table 5.25.
Published 3rd August 2019. Last reviewed 30th December 2021.
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