Normally, for basket and paddle apparatus, the volume of the dissolution medium is 500 mL to 1000 mL, with 900 mL as the most common volume. The volume can be raised to between 2 and 4 L, using larger vessels and depending on the concentration and sink conditions of the drug; justification for this procedure is expected.

The significance of deaeration of the medium should be determined, because air bubbles can interfere with the test results, acting as a barrier to dissolution if present on the dosage unit or basket mesh. Further, bubbles can cause particles to cling to the apparatus and vessel walls. On the other hand, bubbles on the dosage unit may increase buoyancy, leading to an increase in the dissolution rate, or may decrease the available surface area, leading to a decrease in the dissolution rate. A dearation method is described as a footnote in the Procedure section under Dissolution 711. Typical steps include heating the medium, filtering, and drawing a vacuum for a short period of time. Other methods of deaeration are available and in routine use throughout the industry. Media containing surfactants are not usually deaerated because the process results in excessive foaming. To determine whether deaeration of the medium is necessary, results from dissolution samples run in nondeaerated medium and deaerated medium should be compared.

The use of enzymes in the dissolution medium is permitted in accordance with Dissolution 711 when dissolution failures occur as a result of cross-linking with gelatin capsules or gelatin-coated products.

An in-depth discussion on IVIVC can be found in In Vitro and In Vivo Evaluation of Dosage Forms 1088. A brief discussion follows.

Biorelevant medium is a medium that has some relevance to the in vivo performance of the dosage unit. Choice of a biorelevant medium is based on (1) a mechanistic approach that considers the absorption site, if known, and (2) whether the rate-limiting step to absorption is the dissolution or permeability of the compound. In some cases, the biorelevant medium will be different from the test conditions chosen for the regulatory test, and the time points are also likely to be different. If the compound dissolves quickly in the stomach and is highly permeable, gastric emptying time may be the rate-limiting step to absorption. In this case, the dissolution test should demonstrate that the drug is released quickly under typical gastric (acidic) conditions. On the other hand, if dissolution occurs primarily in the intestinal tract (e.g., for a poorly soluble, weak acid), a higher pH range (e.g., simulated intestinal fluid with a pH of 6.8) may be more appropriate. The fed and fasted states may also have significant effects on the absorption or solubility of a compound. Compositions of media that simulate the fed and fasted states can be found in the literature. These media reflect changes in pH, bile concentrations, and osmolarity after meal intake and therefore have a composition different from that of typical compendial media. They are primarily used to establish in vitro–in vivo correlations during formulation development and to assess potential food effects and are not intended for quality control purposes. For quality control purposes, the substitution of natural surfactants (bile components) with appropriate synthetic surfactants is permitted and encouraged because of the expense of the natural substances and the labor-intensive preparation of the biorelevant media.

The choice of apparatus is based on knowledge of the formulation design and the practical aspects of dosage form performance in the in vitro test system. For solid oral dosage forms, Apparatus 1 and Apparatus 2 are used most frequently.

When Apparatus 1 or 2 is not appropriate, another official apparatus may be used. Apparatus 3 (Reciprocating Cylinder) has been found to be especially useful for bead-type modified-release dosage forms. Apparatus 4 (Flow-Through Cell) may offer advantages for modified-release dosage forms that contain active ingredients with limited solubility. In addition, Apparatus 3 or Apparatus 4 may have utility for soft gelatin capsules, bead products, suppositories, or poorly soluble drugs. Apparatus 5 (Paddle over Disk) and Apparatus 6 (Rotating Cylinder) have been shown to be useful for evaluating and testing transdermal dosage forms. Apparatus 7 (Reciprocating Holder) has been shown to have application to nondisintegrating oral modified-release dosage forms, as well as to transdermal dosage forms.

Some changes can be made to the apparatus; for example, a basket mesh size other than the typical 40-mesh basket (e.g., 10, 20, 80 mesh) may be used when the need is clearly documented by supporting data. In countries where available mesh sizes vary from the USP-specified mesh value, basket material with the nearest metric dimension should be used. Care must be taken that baskets are uniform and meet the dimensional requirements specified under Dissolution 711. If the basket screens become clogged during dissolution of capsule or tablet formulations, it may be advisable to switch to the paddle method. The volume can be increased from the typical 900 to 1000 mL by using 2- and 4-L vessels to assist in meeting sink conditions for poorly soluble drugs.

A noncompendial apparatus may have some utility with proper justification, qualification, and documentation of superiority over the standard equipment. For example, a small-volume apparatus with mini paddles and baskets may be considered for low-dosage strength products. The rotating bottle or static tubes (jacketed stationary tubes enclosed with a water jacket and equipped with a magnetic stirrer) may also have utility for microspheres and implants, peak vessels for eliminating coning, and modified flow-through cells for special dosage forms, including powders and stents.

When sinkers are used, a detailed description of the sinker must be stated in the written procedure. It may be useful to evaluate different sinkers, recognizing that sinkers can significantly influence the dissolution profile of a dosage unit. When transferring the procedure, the sinkers should be duplicated as closely as possible in the next facility. There are several types of commercially available sinkers. A method for making sinkers by hand, sinkers that are similar to “a few turns of wire helix” as described in Apparatus 2 (Paddle Apparatus) under Dissolution 711, is described below.

Capsule Shell Type	Length of Wire (cm)	Diameter Size (cm)	Cork Bore Number
#0, elongated	12	0.8	4
#1 and #2	10	0.7	3
#3 and #4	8	0.55	2

For immediate-release capsule or tablet formulations, Apparatus 1 (baskets) at 100 rpm or Apparatus 2 (paddles) at 50 or 75 rpm are most commonly used. Other agitation speeds and apparatus are acceptable with appropriate justification.

Rates outside 25 to 150 rpm are usually inappropriate because of the inconsistency of hydrodynamics below 25 rpm and because of turbulence above 150 rpm. Agitation rates between 25 and 50 rpm are generally acceptable for suspensions. For dosage forms that exhibit coning (mounding) under the paddle at 50 rpm, the coning can be reduced by increasing the paddle speed to 75 rpm, thus reducing the artifact and improving the data. If justified, 100 rpm may be used, especially for extended-release products. Decreasing or increasing the apparatus rotation speed may be justified if the profiles better reflect in vivo performance and/or the method results in better discrimination without adversely affecting method reproducibility.

Selection of the agitation and other study design elements for modified-release dosage forms is similar to that for immediate-release products. These elements should conform to the requirements and specifications given in Dissolution 711 when the apparatus has been appropriately calibrated.

For immediate-release dosage forms, the duration of the procedure is typically 30 to 60 minutes; in most cases, a single time point specification is adequate for Pharmacopeial purposes. Industrial and regulatory concepts of product comparability and performance may require additional time points, which may also be required for product registration or approval. A sufficient number of time points should be selected to adequately characterize the ascending and plateau phases of the dissolution curve. According to the Biopharmaceutics Classification System referred to in several FDA Guidances, highly soluble, highly permeable drugs formulated with rapidly dissolving products need not be subjected to a profile comparison if they can be shown to release 85% or more of the active drug substance within 15 minutes. For these types of products a one-point test will suffice. However, most products do not fall into this category. Dissolution profiles of immediate-release products typically show a gradual increase reaching 85% to 100% at about 30 to 45 minutes. Thus, dissolution time points in the range of 15, 20, 30, 45, and 60 minutes are usual for most immediate-release products. For rapidly dissolving products, including suspensions, useful information may be obtained from earlier points, e.g., 5 to 10 minutes. For slower-dissolving products, time points later than 60 minutes may be useful. Dissolution test times for compendial tests are usually established on the basis of an evaluation of the dissolution profile data.

So-called infinity points can be useful during development studies. To obtain an infinity point, the paddle or basket speed is increased at the end of the run for a sustained period (typically 15 to 60 minutes), after which time an additional sample is taken. Although there is no requirement for 100% dissolution in the profile, the infinity point can provide data that may supplement content uniformity data and may provide useful information about formulation characteristics during initial development or about method bias.

For an extended-release dosage form, at least three test time points are chosen to characterize the in vitro drug release profile for Pharmacopeial purposes. Additional sampling times may be required for drug approval purposes. An early time point, usually 1 to 2 hours, is chosen to show that there is little probability of dose dumping. An intermediate time point is chosen to define the in vitro release profile of the dosage form, and a final time point is chosen to show the essentially complete release of the drug. Test times and specifications are usually established on the basis of an evaluation of drug release profile data. For products containing more than a single active ingredient, drug release is to be determined for each active ingredient.

Visual observations and recordings of product dissolution and disintegration behavior are very useful because dissolution and disintegration patterns can be indicative of variables in the formulation or manufacturing process. To accomplish visual observation, proper lighting (with appropriate consideration of photodegradation) of the vessel contents and clear visibility in the bath are essential. Documenting observations by drawing sketches and taking photographs or videos can be instructive and helpful for those who are not able to observe the real time dissolution test. Observations are especially useful during method development and formulation optimization. Examples of typical observations include, but are not limited to, the following:

There are many brands of autosamplers, including semiautomated and fully automated systems. Routine performance checks, cleaning, and maintenance as described in the pertinent standard operating procedures or metrology documents are useful for reliable operation of these devices.

Some instruments are equipped with sampling through the basket or paddle shaft. Proper validation (e.g., demonstrated equivalence to results with the usual sampling procedure) may be required.

The disturbance of the hydrodynamics of the vessel by sampling probes should be considered and adequate validation performed to ensure that the probes are not introducing a significant change in the dissolution rate.

Comparison of manual and automated procedures should be performed to evaluate the interchangeability of the procedures. This can be accomplished by comparing data from separate runs or, in some cases, by sampling both ways from the same vessel. Results should be consistent with the requirements for intermediate precision (described in this chapter in Validation) if the procedures are to be considered interchangeable.

Other aspects of automation validation may include carryover of residual drug, effect of an in-residence probe (simultaneous sampling as mentioned above may not be suitable in this case), adsorption of drug, and cleaning and/or rinse cycles.

Filtration of the dissolution samples is usually necessary to prevent undissolved drug particles from entering the analytical sample and further dissolving. Also, filtration removes insoluble excipients that may otherwise cause high background or turbidity. Prewetting of the filter with the medium may be necessary.

Filters can be in-line or at the end of the sampling probe or both. The pore size can range from 0.45 to 70 µm. The usual types of filters are depth, disk, and flow-through. However, if the excipient interference is high, if the filtrate has a cloudy appearance, or if the filter becomes clogged, an alternative type of filter or pore size should be evaluated.

Adsorption of the drug(s) onto the filter needs to be evaluated. If drug adsorption occurs, the amount of initial filtrate discarded may need to be increased. If results are still unsuitable, an alternative filter material may be sought.

Filter validation may be accomplished by preparing a suitable standard solution or a completely dissolved sample solution (e.g., prepared as a typical sample in a vessel or a sample put in a beaker and stirred with a magnetic stirrer for 1 hour). For standard solutions, compare the results for filtered solutions (after discarding the appropriate volume) to those for the unfiltered solutions. For sample solutions, compare the results for filtered solutions (after discarding the appropriate volume) to those for centrifuged, unfiltered solutions.

Centrifugation of samples is not preferred, because dissolution can continue to occur and because there may be a concentration gradient in the supernatant. A possible exception might be for compounds that adsorb onto all common filters.

It is necessary to demonstrate that the results are not unduly affected by placebo constituents, other active drugs, or degradates.

The placebo consists of all the excipients and coatings (inks, sinker, and capsule shell are also included when appropriate) without the active ingredient. Placebo interference may be determined by weighing samples of the placebo blend and dissolving or dispersing them in dissolution medium at concentrations that would be encountered during testing. It may be desirable to perform this experiment at 37 by comparing it to the 100% standard by the formula:

in which C is the concentration, in mg per mL, of the standard; AP and AS are the absorbances of the placebo and the standard, respectively; V is the volume, in mL, of the medium; and L is the label claim, in mg. The interference should not exceed 2%.

If the placebo interference exceeds 2%, then method modification—such as (1) choosing another wavelength, (2) baseline subtraction using a longer wavelength, or (3) using HPLC—may be necessary in order to avoid the interference. When other active drugs or significant levels of degradates are present, it is necessary to demonstrate that these do not significantly affect the results. One procedure for doing this is to measure the matrix in the presence and absence of the other active drug or degradate: any interference should not exceed 2%.

Linearity and range are typically established by preparing solutions of the drug, ranging in concentration from below the lowest expected concentration to above the highest concentration during release. This may be done in conjunction with accuracy/recovery determination. The scheme may be altered if different flow-cell sizes or injection volumes are used.

Typically, solutions are made from a common stock if possible. For the highest concentration, the determination may not exceed the linearity limits of the instrument.

Organic solvents may be used to enhance drug solubility for the preparation of the standard solutions; however, no more than 5% (v/v) of organic solvent in the final solution should be used, unless validated.

Linearity is typically calculated by using an appropriate least-squares regression program. Typically, a square of the correlation coefficient (r2 0.98) demonstrates linearity. In addition, the y-intercept must not be significantly different from zero.

Accuracy/recovery are typically established by preparing multiple samples containing the drug and any other constituents present in the dosage form (e.g., excipients, coating materials, capsule shell) ranging in concentration from below the lowest expected concentration to above the highest concentration during release.

In cases of poor drug solubility, it may be appropriate to prepare a stock solution by dissolving the drug substance in a small amount of organic solvent (typically not exceeding 5%) and diluting to the final concentration with dissolution medium. An amount of stock solution equivalent to the targeted label claim may be added to the vessel instead of the drug powder. Similarly, for very low strengths, it may be more appropriate to prepare a stock solution than to attempt to weigh very small amounts. The measured recovery is typically 95% to 105% of the amount added. Bracketing or matrixing of multiple strengths may be useful.

A special case for validation is the Acid Stage procedure described in Delayed-Release Dosage Forms under Dissolution 711. The limit of not more than 10% needs to be validated. If the compound degrades in acid, the validation experiment must address this fact.

The evaluation of robustness, which assesses the effect of making small, deliberate changes to the dissolution conditions, typically is done later in the development of the drug product. The number of replicates (typically 3 or 6) is dependent on the intermediate precision.

Parameters to be varied are dependent on the dissolution procedure and analysis type. They may include medium composition (e.g., buffer or surfactant concentration), pH, volume, agitation rate, and temperature. For HPLC analysis, parameters may include mobile phase composition (percentage organic, buffer concentration, pH), flow rate, wavelength, column temperature, and multiple columns (of the same type). For spectrophotometric analysis, the wavelength may be varied.

The standard solution is stored under conditions that ensure stability. The stability of the standard is analyzed over a specified period of time, using a freshly prepared standard solution at each time interval for comparison. The acceptable range for standard solution stability is typically between 98% and 102%.

The sample solution is typically stored at room temperature. The sample is analyzed over a specified period of time using the original sample solution response for comparison. The typical acceptable range for sample solution stability may be between 98% and 102% compared with the initial analysis of the sample solutions. If the solution is not stable, aspects to consider could be temperature (refrigeration may be needed), light protection, and container material (plastic or glass).

The procedure may state that the standards and samples need to be analyzed within a time period demonstrating acceptable standard and sample solution stability.

Samples may be automatically introduced into the spectrophotometer using autosippers and flow cells. Routine performance checks, cleaning, and maintenance as described in the standard operating procedures or metrology documents are useful for reliable operation of these instruments. Cells with path lengths ranging from 0.02 to 1 cm are typically used. Cell alignment and air bubbles could be sources of error. The smaller path length cells are used to avoid diluting the sample; however, acceptable linearity and standard error need to be demonstrated.

During analysis, standard solutions are typically prepared and analyzed at just one concentration at 100% (or the selected Q value) of the dosage strength. During profile analysis, other concentrations may be useful. A typical blank, standard, and sample may be analyzed in a sequence that brackets the sample with standards and blanks, especially at the beginning and end of the analysis.

In most cases, the mean absorbance of the dissolution medium blank may not exceed 1% of the standard. Values higher than 1% must be evaluated on a case-by-case basis. The typical RSD for UV analysis is usually not more than 2%.

The absorptivity is calculated by dividing the mean standard absorbance by the concentration, in mg per mL, divided by the flow-cell path length in cm. After enough historical data are accumulated, an acceptable absorptivity range for the analyte (using the appropriate flow cell) may be determined. This value may be useful in troubleshooting aberrant data.

Fiber optics as a sampling and determinative method, with proper validation, is an option.

It may be useful to examine the UV spectrum of the drug in solution to select the optimum wavelength.

For HPLC analysis, the compatibility of dissolution media and mobile phase may be examined, especially if large injector volumes (over 100 µL) are needed. Samples are normally analyzed with HPLC using a spectrophotometric detector and an auto-injector. Single injections of each vessel time point with standards throughout the run constitute a typical run design. System suitability tests include, at a minimum, the retention window and injection precision. Typically, the repeatability of an HPLC analysis should be less than or equal to 2% RSD for five or six standard determinations. The standard level is typically at the 100% label claim level, especially for a single-point analysis.

Preparation of the placebo samples for the HPLC analysis is to be performed in the same way as in the spectrophotometric analysis. Examine the chromatogram for peaks eluting at the same retention time as the drug. If there are extraneous peaks, inject the standard solution, and compare retention times. If the retention times are too close, spike the placebo solution with the drug. Chromatograms may also be obtained over an extended run time using the blank (dissolution medium), standard, and sample solution to identify late eluters that may interfere with subsequent analyses.

The validation documentation may include overlaid representative chromatograms or spectra of blank dissolution medium, a filtered placebo solution, a standard solution, and a filtered dissolution sample. Absence of interfering peaks in the placebo chromatogram or lack of absorbance by the placebo at the analytical wavelength demonstrates specificity.