Primera etapa: Clasificación de líneas por pendiente y cuadrante

3.3.1 Preparation of Dissolution Media

Dissolution media were prepared by weighing the individual buffer components into plastic weighing boats prior to their transfer into a clean, 5L volumetric flask. A quantity of distilled water obtained from a Whatman still (Whatman Lab Sales Ltd., Maidstone, Kent, UK) was added to the flask and the contents allowed to mix for 20- 30 minutes until the solid material had fully dissolved. A further amount of distilled water was added in order to produce a final volume of 5L. The pH of the final solution was checked using a pH meter, and when necessary adjusted by the addition of small quantities of HCl or NaOH. Prior to dissolution the required amount of buffer was decanted from a 20L glass storage vessel into a smaller vessel and de-gassed for 20 minutes using a helium purge. This final stage is important since dissolution may be affected by the presence of air bubbles on the product which will effectively decrease the available surface area for dissolution (Nicklasson, 1993).

3.3.2 Preparation of Calibration Standards

It is necessary to prepare calibration standards in order to confirm that there is a linear relationship between, absorbance and drug concentration over the required range, calculate the required amount of material for each run, and determine the wavelength of maximum absorbence (Xmax). A set of calibration standards were prepared for each media. A preliminary investigation was carried out in order to assess the most acceptable and reproducible method of standard preparation.

Method ‘A’ involved adding buffer directly to dry cefuroxime axetil producing a solution of the drug. Approximately 150mg of drug was accurately weighed out into a tarred plastic weighing boat then transferred into a clean, dry IL volumetric flask.

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Approximately 500ml of buffer was added to the flask which was then inverted several times before being placed into an ultrasonic bath for 5-10 minutes. If on visual inspection drug particles were still visible within the solution, the latter process was repeated prior to the solution being made up to volume. Once a suitable solution of the drug had been obtained 1ml aliquots of the stock solution were transferred into

separate 25, 50 and 100ml volumetric flasks and made up to volume with buffer to produce drug concentrations of 6x1 O '*, 3x1 O''* and 1.5xlO '*%w/v respectively. A 1ml aliquot of the 6xlO '*%w/v solution was transferred into a 1 0ml volumetric flask to

produce a final concentration of 6x l0'^%w/v.

After preparing the calibration standards by the above method it was noted that at times extensive sonification was required to ensure the production of a homogeneous solution of the drug. Since it was not possible to standardise these sonification times an alternative method of preparation was investigated. Method ‘B’ involved the initial step of dissolving the weighed drug substance in 2 0ml of methanol prior to adding the

buffer. In this instance the drug dissolved immediately and, on the addition of buffer, only 1 -2 minutes of sonification was required to ensure adequate mixing of the two

solutions. Having selected an appropriate method it was necessary to make a small change to the way in which the stock solution was prepared and diluted. Due to the operating absorbance of the dissolution programme ranging between 0 and 1,

standards which gave equivalent values were required in order to confirm linearity between concentration and absorbance over this range. Approximately 150mg of cefuroxime axetil was accurately weighed out and transferred into a 500ml volumetric flask as opposed to a IL flask. 1ml aliquots of the stock solution were transferred into separate 100, 50, 25 and 10ml volumetric flasks to produce drug concentrations of 3x10 '*, 6x10 '*, 1.2xl0'^and 3xlO'^%w/v respectively. The fifth and final dilution was made by transferring 2ml of stock solution into a 25ml flask producing a drug concentration of 2.4x10'^%w/v.

3.3.3 Construction of Beer Lambert Plots

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the relationship between absorbance and drug concentration. Measurements were made using a Perkin-Elmer 554 UV-VIS Spectrophotometer (Beaconsfield, UK). In order to measure levels due to each concentration it was first necessary to determine the wavelength of maximum absorbance, Xmax-

An initial scan was performed from 900 to lOOnm with both spectrophotometer cells containing buffer in order to check whether or not the buffer itself showed any absorbence over the selected wavelengths. A second scan was then performed in which the front cell contained one of the calibration standards. In all cases this scan produced a single peak around 281nm. The apex of this peak was defined by manually entering wavelengths and recording the corresponding values. The wavelength of maximum absorbance was entered into the spectrophotometer and the machine re­ zeroed with both cells containing buffer. Values were then measured for each of the calibration standards. Cells were rinsed out with buffer between each measurement and the outside wiped with a tissue to remove any excess liquid.

3.3.4 Dissolution Method

Dissolution studies were performed using a six station PHARMA TEST Type PTWS dissolution bath (Pharma Test Apparatebau GmbH, Hainbury, Germany) connected to a Philips PU 8620 UV/VIS/NIR spectrophotometer (Pye Unicam Ltd., Cambridge, UK). The complete dissolution process was automated using a Philips PU 8620 Tablet Dissolution Monitoring System (Pye Unicam, Ltd., Cambridge, UK) connected to a PC. Each of the six dissolution vessels contained 900ml of degassed dissolution medium maintained at 37±0.5°C, and a paddle rotation speed of lOOrpm was used throughout the studies.

Prior to the start of each dissolution run the relevant parameters with respect to the buffer under investigation were entered into the dissolution program. The three fundamental values were, the wavelength of maximum jabsorbance, the weight of active ingredient, i.e. drug substance in each vessel, and the concentration of drug (mg/L) which would give a value of one. As discussed in the previous section, individual calibration curves were constructed for each buffer and it is from these that

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the above values were calculated. From each individual plot the concentration of drug (mg/L) which produced an absorbance of one was determined. This value was then used to calculate the amount of cefuroxime axetil, and in turn SACA which needed to be added to each individual dissolution vessel. An example using Sorensens pH 7.0 is given in Appendix III. Individual dissolution media had a small influence on the weight of material calculated for use in each run, for SACA the mean was

I49.93±3.86mg and for cefuroxime axetil22.38±0.56mg. Finally, the times at which the

drug content was to be analysed were entered along with a sample delay time of 10

seconds which allowed for the sequential transfer of material into each individual dissolution vessel at the start of the run.

The Phillips spectrophotometer contained a total of eight flow through cells. Cells one to six were connected to their respective dissolution vessel, cell seven was isolated containing only buffer and cell eight was not active. Once the instrument was manually zeroed using cell seven it became fully automated and began to measure zero absorbance values for each dissolution vessel. Once completed the program requested the transfer of material and the dissolution run proceeded.

Due to the hydrophobic nature of the stearic acid initial problems were encountered due to inadequate wetting of the material. In order to minimise this it was necessary to wet the material before its transference into the dissolution vessel. This was achieved by initially weighing the material into a clean, glass sample jar then adding 1 0ml of

pre-warmed buffer from the respective dissolution vessel. The jar was when shaken vigorously to ensure adequate wetting of the material and its contents immediately tipped back into the vessel. A previously withdrawn second 10ml aliquot of buffer was then used to rinse the jar, all washings were transferred into the vessel. Data analysis was carried out by the system.

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