Evaluation of Liquid Orals: A solution is a liquid preparation that contains one or more soluble chemical substances dissolved in a specified solvent.
Non-Sterile Liquid Dosage Forms
1. Evaluation Tests for Syrups:
A concentrated solution of a sugar, such as sucrose, in water or other aqueous liquid, sometimes with a medicinal agent added; usually used as a flavored vehicle for drugs. It is commonly expanded to include any liquid dosage form (e.g., oral suspension) in a sweet and viscid vehicle.
Following tests are carried out for the evaluation of syrups:
(a) Transmittance of light: A light transmittance meter is a newer tool that is used to check syrup color. In a light transmittance meter, a syrup sample is checked for color by passing light through the sample. The percent of light transmission is compared to light transmission rates set for different grades. When using one, you need to be sure there are no fingerprints on the syrup test bottle, and that the syrup sample has no bubbles or cloudiness. Any of these conditions may diminish the light that is transmitted through the sample and therefore lowers the grade of the sample.
(b) Visual inspection: With a visual inspection, the ingredients and the final products are carefully examined for purity and appearance. The physical appearance of products for patient adherence and compliance is critical so it should be
- Good looking
- Elegance in appearance
(c) pH measurement: The measurement and maintenance of pH is also a very important step in quality control testing. Generally, there are two different types of methods used in the measurement of pH.
Methods for pH measurement:
- The simplest and cheapest is to dip a piece of pH paper into the sample. The paper is impregnated with chemicals that change color and the color may be compared to a chart supplied with the paper to give the pH of the sample.
- If the greatest accuracy is required a pH meter should be used. A typical pH meter consists of a special measuring glass electrode connected to an electronic meter that measures and displays the pH reading.
(d) Sucrose concentration: The determination of sucrose concentrations is also very important in quality control testing of syrups. If the concentration of sucrose in the syrup is very high it may crystallize the syrup and fewer sucrose concentrations give favor for the microbial growth.
There is no specific method for the determination of sucrose in syrup, HPLC and UVspectroscopy for this purpose are used.
(e) Physical stability in syrups: The syrups must be stable physically.
- Its appearance (no crystallization and microbial growth)
- Colour must be completely soluble with other ingredients
- Odor and taste(palatable).
- The solid material is completely miscible in liquid
2. Evaluation of Elixirs:
Definition: Elixirs are clear, sweetened hydro-alcoholic solutions intended for oral use and are usually flavored to enhance their palatability.
(a) Determination of alcohol content: Elixir usually contains 5 to 40% alcohol. The determination of alcohol unless otherwise specified in the individual monograph. It is suitable for examining most fluid extracts, tinctures and elixirs provided the capacity of the distilling flask is sufficient (commonly two to four times the volume of the liquid to be heated) and the rate of distillation is such that clear distillates are produced. Cloudy distillates may be clarified by agitation with talc, or with calcium carbonate. And filtration is done. After which the temperature of the filtrate is adjusted and the alcohol content determined from the specific gravity. During all manipulations, take precautions to minimize the loss of alcohol by evaporation. For liquids, it is presumed to contain less than 30% of alcohol.
(b) Viscosity measurement: Viscosity is a property of liquids that is directly related to the resistance to flow. Viscosity measurement is a very important quality control test in the case of syrups and elixirs. Viscosity and consistency directly relate to the stability of solutions. If viscosity increases, then there is a chance of increased instability.
3. Evaluation of Suspensions:
A pharmaceutical suspension is a coarse dispersion in which insoluble particles, generally greater than 1 µm in diameter, are dispersed in a liquid medium, usually aqueous.
Following tests are carried out for the evaluation of suspensions:
(a) Sedimentation method: Two parameters are studied for the determination of sedimentation. They are (i) Sedimentation volume and (ii) Degree of flocculation.
(i) Sedimentation Volume: The suspension formulation (50 ml) is poured separately into 100 ml measuring cylinders and sedimentation volume is read after 1, 2, 3, and 7 days, and thereafter at weekly intervals for 12 weeks. Triplicate results are obtained for each formulation. Sedimentation volume is calculated according to the equation:
(ii) Degree of flocculation (β): It is the ratio of the sedimentation volume of the flocculated suspension (F), to the sedimentation volume of the deflocculated suspension, (F∞).
(b) Rheological method: Viscosity of suspensions is of great importance for stability and pourability of suspensions. As we know suspensions have the least physical stability amongst all dosage forms due to sedimentation and cake formation. So as the viscosity of the dispersion medium increases, the terminal settling velocity decreases thus the dispersed phase settle at a slower rate and they remain dispersed for a longer time yielding higher stability to the suspension. On the other hand as the viscosity of the suspension increases, its pourability decreases, and inconvenience to the patients for dosing increases. Thus, the viscosity of suspension should be maintained within the optimum range to yield stable and easily pourable suspensions.
- A practical theological method involves the use of a Brookfield viscometer mounted on a helipad stand. The T-bar spindle is made to descend slowly into the suspension, and the dial reading on the viscometer is then a measure of the resistance the spindle meets at various levels in sediment.
- Data obtained on samples variously aged and stored indicate whether undesired changes are taking place. This measurement is made on undisturbed samples of different ages. The results indicate how the particles are settling with time.
- In the screening study, the better suspensions show a lesser rate of dial reading with spindle turns, i.e., the curve is horizontal for a longer period.
(c) Electrokinetic method: In this zeta potential is measured by using micro electrophoresis apparatus and zeta plus (Brookhaven instruments corporation, USA). It shows the stability of a dispersed system.
E.g. micro-electrophoresis apparatus MK-1.
Zeta potential: The zeta potential of the formulated suspensions is determined using a zeta plus (Brookhaven instruments corporation, USA). Approximately 1 ml of suspension is transferred into a plastic cuvette using a pipette and diluted with distilled water. The Brookhaven zeta potential software is used for the measurement. Parameters set to a temperature of 25o C and refractive index (1.33). The zeta potential of the formulations is determined on days 0, 7, 14, 21, and day 28 post formulation.
(d) Micromeritic method: The stability of suspension depends on the particle size of the dispersed phase. Change in the particle size concerning time will provide useful information regarding the stability of a suspension. A change in particle size distribution and crystal habit can be studied by microscopy and the Coulter counter method.
Photomicroscopy method: The microscope can be used to estimate and detect changes in particle size distribution and crystal form. Rapid processing of photomicrographs is enhanced by attaching a Polaroid camera to the piece of the monomolecular microscope. By using these photomicrographs we can determine the changes in physical properties and stability of suspensions.
(e) Freeze-thaw test: Freeze-thaw test conducted by placing the sample in a freezer for 18 hours followed by thawing at room temperature for 4 to 6 hours. Repeat the freeze-thaw cycle 10 times. This test is conducted to determine the tendency to crystallize or color.
(f) pH measurement: The measurement and maintenance of pH is also a very important step in quality control testing. Generally, there are two different types of methods used in the measurement of pH.
Methods for pH measurement: The simplest and cheapest is to dip a piece of pH paper into the sample.
(g) Visual inspection: With a visual inspection, the ingredients and the final products are carefully examined for purity and appearance. The physical appearance of products for patient adherence and compliance is critical so it should be:
- Good looking
- Elegance in appearance
4. Evaluation of Emulsions:
An emulsion is a system consisting of two immiscible liquid phases, one of which is dispersed throughout the other in the form of fine droplets. A third component, the emulsifying agent, is necessary to stabilize the emulsion.
Following are tests carried out for evaluation of emulsions:
(a) Determination of particle size and particle count: Determination of changes in the average particle size or the size distribution of droplets is an important parameter used for the evaluation of emulsions. It is performed by optical microscopy, sedimentation by using Andreason apparatus and colter apparatus.
(b) Determination of viscosity: Determination of viscosity is done to assess the changes that might take place during aging. Emulsions exhibit the non-Newtonian type of flow characteristics. The viscometer which should be used maybe a cone and plate viscometer.
(c) Determination of phase separation: This is another parameter used for assessing the stability of the formulation. Phase separation may be observed visually or by measuring the volume of the separated phases.
(d) Determination of electrophoretic properties: Determination of electrophoretic properties like zeta potential is useful for assessing flocculation since electrical charges on particles influence the rate of flocculation. Oil in water emulsion having a fine particle size will exhibit low resistance but if the particle size increase, then it indicates a sign of oil droplet aggregation and instability.
(e) Electrical conductivity: It is determined by using platinum electrodes (diameter 0.4 mm, distance 4mm) micro amperometrically to produce a current of 15 to 50 mA. Measurements are made on emulsions stored at room temperature or 37o C for short time. Stable o/w emulsion offers less resistance, but droplet aggregation increases resistance. A stable w/o emulsion does not conduct electrodes, but with the droplet, coagulation conductivity increases.
Sterile Dosage Forms
1. Evaluation of Parenterals:
Following tests are carried out for the evaluation of parenteral:
(a) Leaker test:
- Leakage occurs when a discontinuity exists in the wall of a package that can allow the passage of gas under the action of pressure or concentration differential existing across the wall.
- The presence of capillary pores or tiny cracks can cause microbes or other dangerous contaminants to enter the ampoules or may lead to the leakage of contents to the outside. This may lead to contamination of the sterile contents and also spoilage of the appearance of the package.
- Changes in temperature during storage can cause expansion and contraction of the ampoule and its contents, thereby accentuating interchange if an opening exists.
- Leaker test for ampoules is intended to detect incompletely sealed ampoules so that they can be discarded to maintain the sterile conditions of the medicines.
- Tip seals are more likely to be incompletely closed than pull seals. Open capillaries or cracks at the point of seal result in leakers.
- Leakers are visibly detected by this process. Ampoules are placed in a vacuum chamber. Completely submerged in a deeply colored dye solution of about 0.5-1% methylene blue.
- A negative pressure is applied within the ampoule. Subsequent atmospheric pressure causes the dye to penetrate on opening thus making it visible after the ampoule has been washed. The vacuum, about 27 inches Hg, should be sharply released after 30 minutes.
- Detection of leakers is prominent when ampoules are immersed in a bath of dye during the autoclaving cycle as this has the advantage of accomplishing both leaker detection and sterilization in one operation.
Result: The color from the dye will be visible within a leaker.
Disadvantages: Capillaries of 15 microns or smaller diameter cannot be detected by this test. Vials and bottles are not subjected to such a leaker test as the rubber closer is not rigid.
(b) Pyrogen test:
(i) LAL bacterial endotoxin test: The LAL (Limulus amebocyte lysate) assay is an in-vitro assay used to detect the presence and concentration of bacterial endotoxins in drugs and biological products.
Endotoxins, which are a type of Pyrogen, are lipopolysaccharides present in the cell walls of gram-negative bacteria.
Pyrogens as a class are fever-inducing substances that can be harmful or even fatal if administered to humans above certain concentrations. Water can be a source of pyrogens, so it may be important to routinely monitor water systems using the bacterial endotoxins test.
Procedure: The solution of endotoxins containing preparation is added to the lysate derive from hemolymph cells of horseshoe crab (Limulus Polyphemus).
The result of the reactions is turbidity or precipitation or gelation of the mixture. This is used as a quantitative measure to estimate the endotoxin content. The rate of reaction depends upon the concentration of endotoxins, pH, temperature, and presence of clotting enzyme and cuttable proteins from the lysate. The quantities of endotoxins are expressed in defined endotoxin units (EU). The endotoxin limit for given test preparation is calculated from the expression k/M; where M is the maximum dose administered to adults per kg/hr. The value for K is 5.0 EU/kg for parenteral preparations and it is 0.2 EU/kg for intrathecal preparations.
(ii) Pyrogen test ‘fever response of rabbit’:
Sham test: It is performed to select the proper animals for the main tests.
Rabbit test: The rabbit Pyrogen test is an in-vivo test to detect pyrogens qualitatively. Rabbits have a similar Pyrogen tolerance to humans, so by observing a change in body temperature in rabbits it is possible to decide the presence of pyrogens. This method can detect non-bacterial endotoxin.
- Withheld food on the day of the experiment.
- Record the initial temperature of the rabbits, any rabbit show temperature of more than 39o C should be excluded.
- Inject the sample into the ear vein of each rabbit.
- Check the temperature after 30 minutes, 1, 2, and 3 hours.
- Biological variation
- Not for antipyretic drug
- The test is positive when each rabbit show increase in temperature.
- If only two of the three rabbits show an increase in temperature, repeat the test using a group of five, and the test will be positive if the four of the five rabbits show an increase in temperature.
(c) Sterility test: Sterility testing attempts to reveal the presence or absence of viable microorganisms in a sample number of containers taken from a batch of products.
Based on results obtained from testing the sample a decision is made as to the sterility of the batch.
The primary official test is performed using filtration but the direct transfer is used if membrane filtration is unsuitable.
(i) Membrane filtration method: Media suitable for sterility tests are:
- Fluid thioglycolate medium
- Soya bean casein digest medium
- Wash the filters with fluids to remove inhibitory properties, cutting the membranes aseptically into equal parts and transferring one of the parts to each type of culture medium used.
The media are then incubated under prescribed conditions.
(ii) Direct inoculation method: This method is only used when membrane filtration is not possible the sample is inoculated directly into the media or the device is placed directly into the media.
Result: If no growth in the media then the test is positive.
(d) Particulate evaluation:
- It has been shown that particles of lint, rubber, insoluble chemicals, and other foreign matter can produce emboli in the vital organs of animals and human beings.
- The USP specifies that good manufacturing practice (GMP) requires that each final container of injection is subjected individually to a visual inspection and that containers in which visible particles can be seen should be discarded.
- Therefore, all of the product units from a production line currently are being inspected individually by human inspectors under good light, baffled against reflection into the eye and against a black-and-white background.
- The USP has identified two test methods.
- The first test to be used is the light obscuration test, which uses an electronic instrument designed to count and measure the size of the particles using a shadow cast by the particle as it passes through a high-intensity light beam.
- If the injection formulation is not a clear, colorless solution, it exceeds the limits specified for the light obscuration test, it is to be subjected to the microscopic count test.
(e) Weight variation or uniformity of content:
- This test is intended for sterile solids used for parenteral preparations.
- The weight of 10 individual sterile units is notes and the content is removed from them and the empty individual sterile unit is weighed in turn.
- Then the content of the active ingredient in each sterile unit is calculated by performing the assay according to the individual monographs.
- Then the net weight is calculated by subtracting empty sterile unit weight from the gross weight. The content in 10 sterile units is calculated by performing the assay.
- The dose uniformity is met if the amount of active ingredient is within the range of 35-115% of the label claim as determined by the content uniformity method or weight variation method.
- The dose uniformity is also met if the potency value is 100% in the individual monograph or less of label claim multiplied by the average of limits specified for potency in individual monograph divided by 100 provided that the relative standard deviation in both the cases is equal to or less than 60%. The fore-mentioned test is carried for 20 more sterile units 14.
- The sterile units meet the requirements if not more than one unit is outside the range of 85-115%, no unit is outside the range of 75-125% and the calculated Relative standard is NMT 7.8%.
2. Evaluation of Eye Drops:
Following tests are carried out for the evaluation of eye drops:
(a) Test for sterility: All the ophthalmic preparations should be sterile i.e., free from any viable organism and its spores. Ophthalmic preparations are tested for their sterility.
The following criteria should be followed while carrying out sterility testing. Two sterile culture media are prepared for the detection of aerobic and anaerobic bacteria and fungi.
Test samples are transferred into test tubes containing a clear medium. If the sample contains microorganisms, then the medium becomes turbid. If the sample is free from microorganisms, then the medium remains clear. The tests should be carried out in aseptic conditions.
(i) Membrane filtration method (Method A): This method is generally followed for the products which can be easily filtered and are carried out in aseptic conditions. The apparatus consists of a sterilized filter unit with a sterile membrane filter of 0.45 µ pore size. A single membrane filter is divided into two halves. The test solution is filtered through the membrane filer. One-half of the membrane is placed in a fluid thioglycollate medium at 30-35°C and the other half is placed in soybean casein digest medium at 20-25°C for 7 days.
Result: If the medium shows no growth, then the sample solution passes the test. If the medium shows growth, then the test is repeated. If the growth is observed again, then the sample solution fails the test.
(ii) Direct inoculation method (Method B): Specified quantity of the solution to be tested is drawn through a sterile syringe or pipette. It is mixed with the medium and incubated for 14 days at a specific temperature. Between the 3rd and 7th day of incubation, a portion of the medium is transferred to a fresh medium, if it shows turbidity, then both the old and fresh media are incubated for 14 days.
Result: If the medium shows no growth, then the sample solution passes the test. If the medium shows growth, then the test is repeated. If growth is observed again, then the sample fails the test.
(b) Test for ocular toxicity and irritation: This test assesses the isotonicity of the preparation.
Procedure: Five albino rabbits are selected, the iridal vessels of whom can be easily observed for toxicity and irritation. Based on the type of dosage form, the medicament is extracted using cottonseed oil or saline. Small quantities of the extract are instilled into one eye of all the rabbits, while the sterile saline solution is instilled into the other eye. After one hour all the rabbits are observed for irritation, swelling, or shrinkage of the eye.
Result: No change in the eye into which the preparation is instilled indicates that the preparation under test is safe for use.
(c) Test for preservative efficacy:
- Cultures of microorganisms like Aspergillus niger, Candida albicans, Escherichia coli, and Pseudomonas aeruginosa, each containing about 10,000 – 10, 00,000 organisms per ml are selected.
- Three to four samples of each preparation are taken in sterile test tubes and inoculated with a few ml of each culture separately.
- They are incubated at 20 – 25°C for 28 days and are observed weekly for the appearance of turbidity.
- No growth of microorganisms indicates that the preservative is effective.
(d) Clarity: The clarity of the formulations before and after gelling is determined by visual examination of the formulations under light alternatively against white and black backgrounds.
(e) pH: The pH of each of the prepared ophthalmic formulations is determined by using a pH meter (Equip-Tronics). The pH meter is calibrated before each use with standard pH 4, 7, and 9.2 buffer solutions.
(f) In-vitro diffusion studies: In-vitro release studies are carried out using chambered donor receiver compartment mode (Franz diffusion cell). The in-vitro release was carried out in formulations with different concentrations of generate using dialysis membrane. The diffusion medium 26 ml of simulated tear fluid stirred at 50 rpm at 37o C ±0.5o C. One end of the diffusion tube is covered by a dialysis membrane. The 1 ml formulation is spread on the dialysis membrane and the membrane is placed such that it just touches the diffusion medium (STF) present in the receptor compartment. The drug samples are withdrawn at the interval of one hour for the period of 8 hrs. from the diffusion medium and analyzed by a UV spectrophotometer at 261 nm using simulated tear fluid as blank.
(g) Determination of viscosity: The specified volume of prepared ophthalmic solution is transferred in sample cell which is placed carefully within the adaptor (Brookfield DV-II + PRO viscometer, Adapter spindle No-18). The water of 25°C is circulated through the jacket of the adaptor. The viscosity values are recorded.
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