USP-NF 1236 - Teste Solubilidade

Printed on: Wed Dec 13 2023, 07:09:35 AM(EST) Status: Currently Official on 13-Dec-2023 DocId: GUID-B21E4B4E-B5EF-4EA6-879E-2D03A2476ABB_3_en-US
Printed by: Dario Silva Official Date: Official as of 01-Dec-2020 Document Type: GENERAL CHAPTER @2023 USPC
Do Not Distribute DOI Ref: f5rie DOI: https://doi.org/10.31003/USPNF_M2248_03_01
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á1236ñ SOLUBILITY MEASUREMENTS

INTRODUCTION
BACKGROUND
Thermodynamic Equilibrium and Solubility
Methods of Estimating Aqueous Solubility
Factors that Affect Solubility and Solubility Measurements
EXPERIMENTAL METHODS
Methods for Determination of Equilibrium Solubility
METHODS FOR DETERMINATION OF APPARENT SOLUBILITY
Intrinsic Determination (Rotating Disk)
Potentiometric Titration
Turbidimetry
Physical Assessment of Solubility
SOLUBILITY MEASUREMENTS IN BIORELEVANT MEDIA
Human Fasted-State Simulated Gastric Fluid (FaSSGF)
Human Fed-State Simulated Gastric Fluid (FeSSGF)
Human Fasted-State Simulated Intestinal Fluid (FaSSIF-V2)
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Human Fed-State Simulated Intestinal Fluid (FeSSIF-V2)
Human Simulated Colonic Fluid—Proximal Colon (SCoF2)
Human Simulated Colonic Fluid—Distal Colon (SCoF1)
Canine Fasted-State Simulated Gastric Fluid (FaSSGFc pH 1.2–2.5)
Canine Fasted-State Simulated Gastric Fluid (FaSSGFc pH 2.5–6.5)
Bovine Simulated Ruminal Fluids

GLOSSARY
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Canine Fasted-State Simulated Intestinal Fluid (FaSSIFc)
REFERENCES
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INTRODUCTION1
Solutes may differ in both the extent and the rate at which they dissolve in a solvent. Solubility is the capacity of the solvent
to dissolve a solute whereas dissolution rate is how quickly the solubility limit is reached. Equilibrium solubility is the
concentration limit, at thermodynamic equilibrium, to which a solute may be uniformly dissolved into a solvent when excess
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solid is present. The apparent solubility may be either higher or lower than the equilibrium solubility due to transient
supersaturation or incomplete dissolution due to insufficient time to reach equilibrium. Equilibrium can be defined as sufficiently
converged when it no longer changes significantly during a certain time frame. Solubility may be stated in units of concentration
such as molality, molarity, mole fraction, mole ratio, weight/volume, or weight/weight.
Solubility can be expressed in absolute as well as relative terms. One method of describing the absolute solubility is the
descriptive solubility defined in General Notices, 5.30 Description and Solubility. Relative measures of the solubility are important
for predicting the drug delivery characteristics of a dosage form and characterizing a drug as either high solubility or low
solubility in the biopharmaceutics classification system (BCS) (1).
Accurate determination of the solubility of pharmaceutical materials is important for understanding both quality control and
drug delivery issues for pharmaceutical formulations. The apparent solubility (see the Glossary) of a material is affected by the
physicochemical properties of the material (e.g., surface area, particle size, crystal form), the properties of the solubility media
(e.g., pH, polarity, surface tension, added surfactants, co-solvents, salts), and the control of the solubility measurement
parameters (e.g., temperature, time, agitation method). Additionally, the apparent solubility may be comprised of the intrinsic
solubility of the uncharged moiety, the solubility of the ionized compound, and the effect of solubilizers and multiple crystal
forms or salt forms. Control of these experimental factors during solubility measurements is key to obtaining accurate, reliable
values for the equilibrium solubility of a material.
This chapter will begin with a discussion of the concepts and equations that are relevant to solubility measurements.
Understanding these relationships is fundamental to accurate evaluation of solubility. This will be followed by a brief description
of typical experimental methods used to assess solubility of pharmaceutical materials. Finally, the use of solubility measurements
to obtain biorelevant solubility (for human products) and species-dependent solubility (for veterinary products) will be
discussed.
1 The terms used in this chapter are defined in the Glossary.
https://online.uspnf.com/uspnf/document/1_GUID-B21E4B4E-B5EF-4EA6-879E-2D03A2476ABB_3_en-US 1/13
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BACKGROUND
Thermodynamic Equilibrium and Solubility

Dissolution of a crystalline solid solute can be modeled by the two-step process of melting the crystal into a pure liquid solute
followed by mixing the liquid solute into the solvent. The Gibbs free energy of mixing determines whether, and to what extent,
two compounds mix to form a homogeneous phase.
ΔGmix = ΔHmix − TΔSmix
ΔGmix = Gibbs free energy of mixing

ΔHmix = enthalpy of mixing; indicates if the mixing is an endothermic or exothermic process
T = temperature in degrees Kelvin
ΔSmix = change in entropy (disorder) that results from mixing
If the change in the Gibbs free energy is negative, the mixing will be thermodynamically favored. When equilibrium is
reached, ΔG will be equal to zero. The enthalpy of mixing is due to the breaking of cohesive interactions (solute–solute, solvent–
solvent) and the creation of adhesive interactions (solute–solvent). In other words:
ΔHmix = HUU + HVV − HUV
ΔHmix = enthalpy of mixing
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U = solute
V = solvent
This enthalpy change is also equivalent to the work done by removing a volume of pure solvent and a volume of pure solute
and exchanging them. The enthalpy change is equal to the newly created interfacial surface energy according to:
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�
γUV = solute-solvent interfacial surface tension

AU = interfacial surface area
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γiV = group-solvent interfacial surface tension for group i
Ai = surface area of group i
This equation also shows how the total surface energy can be broken into i smaller groups each with its own surface area,
Ai, and corresponding group-water interfacial tension γiV.
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For an ideal system, ΔHmix is zero because the interactions between the ideal solute and ideal solvent are identical. The entropy
of mixing for an ideal solution always increases as a result of mixing and is given by:
ideal
Δ�mix = R(XU lnXU + XV lnXV)
which under dilute conditions can be simplified to:

ideal
Δ�mix = R lnXU
For an ideal system under dilute conditions when a crystalline solid is in equilibrium with a saturated solution of the same
solute:
ideal ideal
Δ�� = −TΔ�� = − RT lnXU
ΔG
m
lnXU =
RT
R = gas constant
XU = concentration of the solute expressed as a mole fraction
ΔGm = the free energy change from melting the crystalline solid
Which illustrates how the solubility of the substance can be correlated with the melting point. For a real solution, the solute
may also affect (reduce) the disorder in the solvent by inducing structure to the solvent. Therefore, for a real solution, this yields:
real ideal
Δ�mix = ΣγiVAi − T(ΣhiAi + Δ�mix )
� �
hi = entropic effect on solvent of group i with area Ai
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This model allows the total solute surface area (AU) to be broken into smaller pieces (∑Ai) and the contribution of these groups
to the enthalpic and entropic contributions to the free energy can be estimated.
Methods of Estimating Aqueous Solubility

Yalkowsky demonstrated (2,3) that a relatively simple general solubility equation (GSE) may be used to empirically estimate
the intrinsic solubility of compounds in water.
logS0 = 0.5 − 0.01(MP − 25) − logKOW
S0 = intrinsic solubility (of the unionized molecule)

MP = melting point of the crystalline solid (in degrees Celsius)
KOW = octanol–water partition coefficient; the temperature of the water is 25°
The GSE indicates that the aqueous solubility will be reduced for compounds with a higher melting point and compounds
with a higher tendency to partition into an oil phase (octanol). The logarithm of the octanol–water partition in the GSE accounts
for the difference between an ideal solution and an aqueous solution due to the enthalpy of mixing (3). The GSE can also be
used to predict the solubility of ionizable compounds by combining it with the Henderson–Hasselbalch equation if the pKa is
known (see Effect of pH).
Use of GSE requires the measurement of the melting point and partition coefficient (and pKa for ionizable compounds). There
are several computer programs that will support the estimation of the partition coefficient and the pKa for compounds based
on structure (4), but this is not the case for melting points. Efforts to develop computational methods to predict aqueous
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solubilities have relied on training sets of molecules to search for correlations with properties that can be more easily predicted
from the structure (e.g., molecular weight, solvent-accessible surface area, number of rotatable bonds, etc.) (5). The success
of these computational approaches is often limited to molecules that are similar to the training set. These calculational methods
are adequate for providing assistance in prescreening synthetic candidates, but are not sufficiently accurate to substitute for
experimental solubility.
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Factors that Affect Solubility and Solubility Measurements
EFFECT OF PH
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The solubility of ionizable acids and bases is pH dependent because the charged species have a higher affinity for the aqueous
environment than the neutral form. The total solubility of the ionizable acid or base is the sum of the intrinsic solubility and the
amount of ionized solute present at that pH. The Henderson–Hasselbalch equation relates the increase in the solubility to the
pH of the solution relative to the pKa (acidic) or pKa (basic) of the ionizable acid or base.
− pH − pK
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� a
pH = pKa + log ; Stot = S0 1 + 10
HA
pKa = −log(Ka)
Ka = acid dissociation constant
[A−] = molar concentration of the acid’s conjugate base
[HA] = molar concentration of the undissociated weak acid
Stot = total solubility of weak acid
S0 = intrinsic solubility of uncharged moiety
� pK − pH
a
pH = pKa + log ; Stot = S0 1 + 10
+
BH
pKa = −log(Ka)
Ka = base dissociation constant
[B] = molar concentration of the base’s conjugate base
[BH+] = molar concentration of the dissociated base
Stot = total solubility of weak base
S0 = intrinsic solubility of uncharged moiety
The Henderson–Hasselbalch equation helps explain the increase in solubility at the first pKa, but is not useful for modeling
the behavior of polyprotic acids over a pH range incorporating additional pKa values. Because ionizable molecules can differ in
the number and type of ionizable groups, it is important to explore solubility across a range of pH values. Figure 1 illustrates
this pH dependence of the solubility for a molecule with two ionization constants of 5.6 and 11.7. The molecule is charged
below pH 5.6 and above 11.7 and is neutral between these two pH values. Where the molecule is unionized, the solubility is
equal to the intrinsic solubility. For the ionized molecule, the solubility increases on a log scale as the pH changes. The formation
of a salt may limit the solubility at a low or high pH (see Figure 1). If the acid used to adjust the pH contributes the counter-ion
for the salt, the common-ion effect will further suppress the solubility as the counter-ion concentration is increased (see Figure
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1). If the salt is dissolved at higher pH, the salt may supersaturate the solution initially, but will eventually precipitate as whatever
solid form has lower solubility at that pH (6).
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Figure 1. Effect of pH on solubility for an ionizable compound. When the molecule is unionized, the solubility is equal to the
intrinsic solubility. For the ionized molecule, the solubility increases on a log scale as the pH changes. The salt solubility limits
the solubility at a low pH. If the acid used to adjust the pH contributes the counter-ion for the salt, the common-ion effect will
further suppress the solubility as the counter-ion concentration is increased (apparent at pH <2 in the figure).
EFFECT OF SALTS AND COUNTER-IONS

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Ionizable compounds can also form salts with an oppositely charged counter-ion (6). In a solution, in the presence of the
charged counter-ion, the solubility product describes this equilibrium reaction as follows:
For the salt dissolution, AnBm(s) → nA+(aq) + mB−(aq)

solubility product, Ksp = [A+]n[B−]m
The maximum solubility of the salt in a solution is also illustrated in Figure 1. As a result of the formation of the salt, the actual
solubility of the charged molecule is seen to plateau (at a pH below the pKa of the drug in this example) instead of continuing
to increase as predicted by the Henderson–Hasselbalch equation. Because the solubility product, Ksp, is a constant, the solubility
of the ionizable moiety may drop even further if the acid being used to adjust the pH increases the concentration of the
oppositely charged counter-ion. The reduction in the solubility of the charged molecule as the counter-ion concentration is
increased is referred to as the common-ion effect (6). This is frequently seen when hydrochloric acid (HCl) is used to reduce
the pH, and the solubility of the chloride salt is reduced due to increasing chloride concentration (e.g., at pH <2). Although not
illustrated in Figure 1, salts may also limit the solubility on the basic side of the plot (e.g., a sodium salt of an acid moiety), and
the common-ion effect may similarly affect the solubility at a high pH when the compound used to adjust the pH has a
common-ion (e.g., sodium hydroxide).
EFFECT OF CO-SOLVENTS
Water is often a poor solvent for many pharmaceutical ingredients, but water is miscible with other solvents that may provide
good solubility for these substances (e.g., ethanol, propylene glycol, polyethylene glycol, etc.). According to the log-linear
model (2), the log S of the solute can generally be linearly interpolated between two miscible co-solvents. This relationship is
illustrated in Figure 2. When this solubility plot is switched to a linear scale, it becomes evident that even low concentrations of
the poor solvent (typically water) in the co-solvent mixture can dramatically reduce the solubility for the solute. For this reason,
solutions containing co-solvents are particularly prone to precipitation when diluted due to the significant change in solubility.
[NOTE—This simple model, as depicted in Figure 2, assumes that maximum solubility occurs at 100% of the good solvent and
this may not be the case for all co-solvent systems.]
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Figure 2. Illustration of the log-linear model of Yalkowsky and co-workers (2). The log of solubility appears to be linear as a
function of the co-solvent fraction. When plotted on a linear scale, it is apparent that the solubility drops exponentially as the
poor solvent is added to the better solvent.
EFFECT OF SURFACTANTS
Surfactants are amphiphiles, which are characterized by polar and nonpolar regions. When placed in water, a surfactant
prefers to reside at the air-water interface and orients its polar region in water and its nonpolar region to the less polar interface
(air). When the air-water interface is saturated with adsorbed surfactant, additional surfactant molecules aggregate into a
spherical micelle with a polar surface and a nonpolar core. This point when micelles form is known as the critical micelle
concentration (CMC). Above the CMC, the number of micelles in a solution increases linearly as the concentration of surfactant
increases. If a pharmaceutical material is able to partition into the micelle, its solubility will increase linearly as the number of
micelles increases (see Figure 3). The CMC for surfactants is dependent on several factors including temperature, ionic
strength, and pH. As an example, the CMC for sodium lauryl sulfate is 6 mM, and the CMC for polysorbate 80 is 0.012 mM,
in pure water at 25°. The solubilization of a molecule by a surfactant can be evaluated based on two descriptors: the molar
solubilization capacity, and the micelle-water partition coefficient. The micelle-water partition coefficient is the ratio of drug
concentration in the micelle to the drug concentration in water for a particular surfactant concentration (7).
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Figure 3. Solubility enhancement by surfactants. Solubilization requires the formulation of micelles. Below the CMC, added
surfactant results in monomers of surfactant dissolved in the solvent and no solubility enhancement occurs. Above the CMC,
the solubility increases linearly. The slope of this linear increase indicates the solubilization capacity of the micelles.
As illustrated in Figure 3, the solubility in the presence of surfactants is the additive combination of the amount dissolved in
the aqueous phase plus the amount solubilized by the micelles. The micelles will be larger than the solute and will diffuse more
slowly than the solute. Drug delivery in the presence of micelles will be due to the absorption of the free drug in solution as
well as drug delivery by micelle-mediated transport (5,6). Therefore, solubilization by surfactants may not result in an
enhancement in drug delivery that is directly proportional to the increase in aqueous solubility (5,6).
EFFECT OF COMPLEXING AGENTS

Complexing agents may form intermolecular complexes with low-solubility materials and enhance the solubility. Aqueous
solubility of nonpolar molecules in the presence of complexing agents is improved as the nonpolar molecules and the nonpolar
region of the complexing agent are sequestered out of water. When this occurs, the aqueous solution can accommodate more
of the nonpolar molecules. Regardless of the ratio of ligand to solute in these complexes (e.g., 1:1, 2:1, 3:1, etc.), the solubility
enhancement is expected to increase as the concentration of the complexing agent increases (8). This is very similar to
solubilization by surfactants except there will not be a minimum concentration of the complexing agent required. Complexes
with high stability constants may bind solutes strongly enough to enhance aqueous stability. Cyclodextrins are often used to
enhance solubility by forming complexes with drug substances.
EFFECT OF SURFACE AREA (DISSOLUTION RATE)

The Noyes–Whitney equation (as expressed by Nernst and Brunner) states:
DA �� − �
∂�
Rate = ∂� = ℎ
C = concentration of the solute in the solvent at time, t
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D = diffusivity of the solute

A = surface area of the solute particles
CS = saturated solubility of the solute
h = thickness of the diffusion layer
In unstirred solutions, the diffusion layer thickness, h, may be large and is primarily affected by the diffusivity of the solute;
however, good mixing may significantly reduce the diffusion layer thickness. For small particles in well-mixed solutions, the
diffusion layer thickness, h, was found (9) to be proportional to the square root of the particle radius. This equation indicates
that the smaller particles will have greater surface area and will dissolve more quickly. In order to reach the equilibrium solubility
as quickly as possible, the surface area should be kept as high as possible (i.e., smaller particles) and the diffusion layer thickness
kept as small as possible (i.e., good mixing) (9).
For spherical particles, the surface area, A, can be expressed as a function of the total mass, M:
6�
For spherical particles, A =
ρ�
ρ = density
d = particle diameter
The dissolution rate of a material will not affect the equilibrium solubility, but will affect how quickly this equilibrium is
achieved.
∂� 6DM
Rate = ∂� = (C − C)
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ρdh S
EFFECT OF SURFACE ENERGY

The surface energy of the particle may affect the solubility. According to the Kelvin equation, smaller particles have higher
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solubility than larger particles due to the effect of the surface energy on the total Gibbs free energy of the system. Typically,
this effect on solubility only becomes significant for particles smaller than 1 micron. The Kelvin equation quantifies this as:
4��
�
ln � =
RTd
0
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S = apparent solubility
S0 = solubility of an infinitely large particle
γ = surface energy of the solute
Vm = molecular volume of the solute
R = gas constant
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T = temperature
d = diameter of the particle
The solubility difference between smaller and larger particles leads to so-called Ostwald ripening in polydisperse suspensions.
The small particles dissolve and result in a solution that is supersaturated relative to the solubility of the larger particles. This
leads to recrystallization on the surface of the larger particles. The larger particles grow in size while the smaller particles dissolve
resulting in an increase in the mean particle size of the suspension (10).
EXPERIMENTAL METHODS
Methods for Determination of Equilibrium Solubility

SATURATION SHAKE-FLASK METHOD
The shake-flask method is based on the phase solubility technique that was developed 40 years ago and is still considered
by most to be the most reliable and widely used method for solubility measurement today (11–18). The shake-flask method
should be used when equilibrium solubility needs to be determined. Other methods may be used to evaluate apparent solubility,
but are not considered suitable for evaluation of true equilibrium solubility.
The solubility medium selected for solubility measurements should be selected to be relevant to the application with efforts
made to control the surfactant type and concentration, the ionic strength of the buffer, and the types of counter-ions present
in the buffer. When the results are intended to predict absorption or bioavailability, it is recommended that one of the
biorelevant media solutions be used (see Solubility Measurements in Biorelevant Media). When the results are intended to support
the development of dissolution tests, it is recommended that the dissolution medium be used. For research purposes, when
the pH dependence of the compound is being evaluated, a buffer that allows control of ionic strength and counter-ion types
over a wide pH range is recommended (e.g., Britton–Robinson or Sörensen buffers). For solubility measurements that will be
used for BCS classification, USP-recommended buffers should be used (19).
Sample preparation: The test substance is typically prepared by adding an excess of solid to the solubility medium, which is
in a stoppered flask or vial. The amount of medium in the flask or vial does not need to be measured accurately. It is
recommended that the solid be added to the solubility medium at about 1–2 mg/mL in excess of the estimated solubility. (For
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low-solubility compounds, a concentration of 1–2 mg/mL may be sufficient.) The surface area of the solid may be increased by
grinding (e.g., in a mortar and pestle) of the sample prior to addition to the medium or by sonication of the sample after
addition to the medium. [CAUTION—It is advised to use caution when employing high-energy methods to increase the surface
area because it may alter the solid form of the solute.] It is recommended that sample preparation be performed in triplicate
to provide at least 3 solubility results for each test condition.
Equilibration of solution: To facilitate dissolution of the solid, the suspension should be actively mixed or agitated. As a good
initial time of incubation, 24 h is recommended; however, the suitability of the selected equilibration time must be verified.
The temperature of the suspension should be well controlled during this dissolution phase (±0.5°). Following the dissolution
phase, it is recommended that the excess solid be allowed to sediment completely. Sedimentation and decantation is
recommended as the safest method for separation of the solid from the saturated solution. For non-clarifying colloid solutions,
centrifugation can be used. Sampling of the supernatant should avoid incorporating any undissolved solid, as this will
significantly affect the solubility result. The transfer pipet needs to be pretreated with sample solution before use, so that surface
adsorption is not altering the transferred solution. If filtration cannot be avoided, then it is essential that the proper filter type
is selected. For polar, ionized species, hydrophobic type filters (nylon) are recommended; while for unionized species the
hydrophilic type filters [e.g., polyvinylidene difluoride (PVDF) or polyethersulfone (PES)] are recommended. The filtration should
be done after sedimentation, and not directly after agitation. Presaturation of the filter is necessary (i.e., the initial portions of
filtrate should be discarded). The temperature of the suspension during the sedimentation and centrifugation steps must also
be well controlled (±0.5°) and be equivalent to the temperature at which solubilization occurred.
Saturation (equilibrium) has been reached when multiple samples, assayed after different equilibration time periods, yield
equivalent results (e.g., change by less than 5% over 24 h, or less than 0.2%/h). To confirm that the apparent solubility is the
equilibrium solubility, it is recommended that the same suspension be re-equilibrated via the same procedure (e.g., mix for an
additional 24 h).
Analysis of solution: The requirements for the analytical method used to quantitate the concentration of the solute and the
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level of analytical validation required should be commensurate with the intended use of the solubility data. In general, the
method should be linear and specific. The supernatant solution may need to be diluted before analysis to be within the linearity
of the analytical method and to avoid possible precipitation. The solution may be analyzed by UV-Vis spectrometry or by liquid
chromatography methods to determine the soluble concentration. The advantage of HPLC is that it can detect instability by
resolving drug-related impurities (13,20).
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It is recommended that the excess solid in the suspension be analyzed at the end of the solubility measurement to verify that
the solid form has not changed. In cases where the solid form has changed, it is likely that the new solid form has a lower
solubility than the initial solid form and that the observed solubility is due to the new lower-solubility form; however, this should
be evaluated on a case-by-case basis. Powder X-ray diffraction (PXRD), Raman or near infrared (NIR) spectrometry, or evaluation
of the melting point by differential scanning calorimetry (DSC) are examples of techniques that can be used to evaluate the
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solid form. Solutes that are unstable (either chemically or physically) during the equilibration time are not suitable for equilibrium
solubility measurements by the shake-flask method. For example, amorphous drugs that will convert to lower solubility salts or
polymorphs should be analyzed using one of the apparent solubility methods.
Reporting of solubility results: If a non-standard composition for the media is used in the solubility determination, the details
of the composition should be reported. The ionic strength of the media used in the solubility determination should be calculated
and reported with the solubility result. The pH of the supernatant solution should be recorded (at the temperature of the
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solubility measurement) when the sample is withdrawn for analysis. When using well-defined, standard media, it is
recommended that the pH of the media not be adjusted to compensate for alteration of the pH by the dissolving species; rather,
the solubility value should be reported at the pH value and temperature observed at the end of the equilibration step (12,18).
If the pH of the media is significantly affected by the dissolving species and solubility at a particular pH is desirable, it is
recommended to perform an additional solubility measurement in a higher buffer-capacity medium. Report the mean
temperature and the precision of temperature control during the equilibration.
The precision of the reported equilibrium solubility should reflect the level of agreement between the measurements rather
than the precision of the solubility analysis. Standard deviations in the measured solubility (based on averaging 3 or more
independent samples) should be included.
Change to read:
METHODS FOR DETERMINATION OF APPARENT SOLUBILITY
Intrinsic Determination (Rotating Disk)

The measurement of intrinsic dissolution is described more fully in ▲Intrinsic Dissolution Testing Procedures for Rotating Disk
and Stationary Disk á1087ñ▲ (CN 1-Dec-2020). This measurement technique may also be used to assess the solubility.
To apply this method to the measurement of solubility, the dissolution experiment must be continued to the point that the
rate of dissolution is insignificant (e.g., less than 5%/24 h or less than 0.2%/h).
All the requirements discussed for the shake flask method in Analysis of solution and Reporting of solubility results will also
apply to measurements using the intrinsic dissolution apparatus.
Potentiometric Titration
The potentiometric acid–base titration for solubility measurements is based on a characteristic shift in the middle of the
titration curve that is caused by precipitation (21). For the titration, accurate volumes of a standardized acid or base are added
to a solution containing an ionizable substance and a salt, for example, 0.15 M potassium chloride (KCl), which is included to
increase the accuracy of the measurements. Sparging (a technique that involves bubbling a chemically inert gas such as
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nitrogen, argon, or helium through a liquid) with argon prevents carbon dioxide (CO2) from the atmosphere from influencing
the pH value. A glass electrode is used to monitor the pH value continuously. The potentiometric titration curve is obtained by
plotting the pH value against the consumed volume of acid/base (21).
Turbidimetry
Turbidimetry involves the dissolution of a compound in an organic solvent, for example, dimethyl sulfoxide (DMSO). The
resulting solution is added to a buffer solution in intervals adequate to characterize changes in turbidity. Further aliquots of the
solution are added after the first detection of turbidity by light scattering. Subsequently, the volume added can be plotted
against the turbidity. The solubility is then estimated by back-extrapolation to the point where precipitation began. This method
can be used to measure as many as 50–300 samples per day. When using solvents such as DMSO, drawbacks include the
increase in solubility of the drug substance for the short duration of the experiment, which leads to a kinetic rather than
thermodynamic solubility, the formation of a supersaturated solution, and the undefined crystalline form of the precipitated
solid (unless it is removed from the suspension and characterized).
Physical Assessment of Solubility

For compounds with extremely high solubility, as well as for biologics and other molecules that lack chromophores or are
not easy to quantitate in solution, a physical assessment of solubility may be used to assess apparent solubility. In this case, the
principle of measurement is the loss of solid material to the solution phase. Equilibrium may be assessed by both the stability
of the weight loss as well as the stability of the change in the physical properties of the resulting solution (e.g., refractive index,
density, osmolality, etc.). Because the physical assessment of solubility does not involve a specific or stability-indicating assay,
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it is recommended that some attempt be made to verify the stability and purity of the solute. Also, evaporation of the solvent
should be carefully monitored and controlled during the solubility measurement performed by this method.
SOLUBILITY MEASUREMENTS IN BIORELEVANT MEDIA (22–26)

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The use of simple aqueous buffers to evaluate the aqueous solubility of a drug substance as a function of pH may
underestimate the bioavailability (22,23). The media recipes presented here are examples that may be used to evaluate the
solubility in simulated human, canine, and bovine (ruminant) fluids to make improved estimates of bioavailability.
The temperature of the solubility medium should be controlled at ±0.5° during biorelevant solubility measurements.
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Biorelevant solubility measurements should follow the shake-flask method, including solubility measurements at multiple time
points to confirm that equilibrium has been achieved. The salt form of a drug added to the solubility medium may significantly
affect the composition of the medium (i.e., ionic strength, pH, etc.). Therefore, the solubility of a salt and a free base of the
same drug should not be assumed to be equivalent unless proven through independent measurements starting with the
different crystalline solids.
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Human Fasted-State Simulated Gastric Fluid (FaSSGF) (24)

The pH is 1.6 at 37°. See Table 1 for the media composition.
Table 1
Concentration Concentration
Ingredient (mM) (g/L)
Hydrochloric acid ~31.3 (q.s. to pH 1.6) ~1.14 (q.s. to pH 1.6)
Sodium chloride 34.2 2.00
Sodium taurocholate 0.08 0.047
Lecithin 0.02 0.015
Pepsin — 0.1
Human Fed-State Simulated Gastric Fluid (FeSSGF) (24)

The pH is 5 at 37°. See Table 2 for the media composition.
Table 2
Hydrochloric acid (q.s. to pH 5) (q.s. to pH 5)
Sodium hydroxide (q.s. to pH 5) (q.s. to pH 5)
Sodium acetate 29.75 2.441
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Table 2 (continued)
Acetic acid 17.12 1.028
Milk, whole 1:1 1:1
Prepare buffer and then mix 1:1 with milk. Adjust to a pH of 5, if necessary.
Human Fasted-State Simulated Intestinal Fluid (FaSSIF-V2) (24)

Table 3
Maleic acid 19.12 2.219
Sodium hydroxide 34.8 1.392
al
Lecithin 0.2 0.165
Human Fed-State Simulated Intestinal Fluid (FeSSIF-V2) (24)

ci Table 4
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Sodium taurocholate 10 5.89

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Lecithin 2.0 1.65
Glyceryl monostearate 5.0 1.79
Sodium oleate 0.8 0.24
Human Simulated Colonic Fluid—Proximal Colon (SCoF2) (25)

Table 5
Sodium hydroxide ~159 (q.s. to pH 5.8) ~5.4 (q.s. to pH 5.8)
Acetic acid, glacial 170 10.2
Human Simulated Colonic Fluid—Distal Colon (SCoF1) (25)

Table 6
Concentration
Ingredient (g/L)
Potassium chloride 0.2
Sodium chloride 8
Potassium phosphate, monobasic 0.24
Sodium phosphate, dibasic 1.44
11
Canine Fasted-State Simulated Gastric Fluid (FaSSGFc pH 1.2–2.5) (26)

Canine gastric pH can vary substantially. Due to interstudy variations in the canine gastric pH estimates, solubility should be
evaluated over a range of 1.2–6.5. The pH of this fluid is adjusted by altering the amount of hydrochloric acid so that pH values
in the range of 1.2–2.5 may be achieved. The pH is 1.2–2.5 at 37°. See Table 7 for the media composition.
Table 7
Ingredient (mM) (mg/mL)
Hydrochloric acid ~3.6–82 (q.s. to pH 1.2–2.5) ~0.13–3 (q.s. to pH 1.2–2.5)
Sodium taurodeoxycholate 0.10 0.054
Lecithin 0.025 0.019
Lysolecithin 0.025 0.012
Canine Fasted-State Simulated Gastric Fluid (FaSSGFc pH 2.5–6.5) (26)
al
Canine gastric pH can vary substantially. Due to interstudy variations in the canine gastric pH estimates, solubility should be
evaluated over a range of 1.2–6.5. The pH of this fluid is adjusted by altering the amount of sodium hydroxide so that pH values
in the range of 2.5–6.5 may be achieved. The pH is 2.5–6.5 at 37°. See Table 8 for the media composition.
Ingredient
ci Table 8
Concentration
(mM)
Concentration
(g/L)
Sodium hydroxide ~14.5–40 (q.s. to pH 2.5–6.5) ~0.58–1.6 (q.s. to pH 2.5–6.5)

ffi
Lecithin 0.025 0.019

O
Canine Fasted-State Simulated Intestinal Fluid (FaSSIFc) (26)

Table 9
Ingredient (mM) (mg/mL)
Sodium dihydrogen phosphate, monohydrate 28.65 3.953
Lecithin 1.25 0.96
Bovine Simulated Ruminal Fluids (27–29)

The media defined here are suitable to represent bovine as well as other ruminant species. The normal pH of a healthy
reticulo-rumen is in the 5.5–6.8 range. High-grain diets typically result in a lower ruminal pH (~5.5), whereas high-forage diets
result in a higher ruminal pH (~6.8).
12
In the abomasum (true stomach), the pH is about 2–3 and is similar to conditions observed in monogastrics and humans.
To represent the abomasum, one may use 0.01 M hydrochloric acid (pH 2), 0.0033 M hydrochloric acid (pH 2.5), or 0.001 M
hydrochloric acid (pH 3). Intestinal pH in ruminants is similar to that observed in monogastrics and humans. The pH at the
pylorus is about 3.0 and increases to about 7.5 in the ileum. To represent the bovine intestinal fluids, one may use one of the
simulated intestinal fluids defined for human or canine above.
The temperature is set for 39°. [NOTE—The pH of media should be confirmed at 39°.] See Table 10 for the media composition.
Table 10
Ingredient High-Grain Diet Simulated Rumen High-Forage Diet Simulated Rumen
Tryptone 2.5 g/L 2.5 g/L
Sodium bicarbonate 4.0 g/L (47.6 mM) 8.75 g/L (104 mM)
Potassium phosphate monobasic anhydrous 1.55 g/L (11.4 mM) 1.55 g/L (11.4 mM)
Sodium phosphate dibasic anhydrous 1.425 g/L (10.0 mM) 1.425 g/L (10.0 mM)
Ammonium bicarbonate 1.0 g/L (12.6 mM) 1.0 g/L (12.6 mM)
Magnesium sulfate heptahydrate 0.15 g/L (0.6 mM) 0.15 g/L (0.6 mM)
Acetic acid 2.40 g/L (40 mM) 1.42 g/L (24 mM)
Propionic acid 2.59 g/L (35 mM) —
Butyric acid 1.32 g/L (15 mM) —
al
Sodium acetate trihydrate — 6.26 g/L (46 mM)
Sodium propionate — 1.44 g/L (15 mM)
Sodium butyrate ci — 1.10 g/L (10 mM)
Micromineral stock solution (see Table 11) 125 µL 125 µL
pH 5.5 6.8
Surface tension ~50 dynes/cm2 ~50 dynes/cm2
Ionic strength 0.102 mol/L 0.240 mol/L

ffi
Buffer capacity 47 mmol/L/pH 49 mmol/L/pH
Table 11
Micromineral Stock Solution Concentration
Ingredient (g/100 mL)
O
Water q.s. to 100 mL
Calcium chloride dihydrate 13.2
Manganese chloride tetrahydrate 10.0
Iron (III) chloride hexahydrate 8.0
GLOSSARY
[NOTE—The following definitions are provided to clarify the use of these terms in the context of this chapter. These definitions
are not intended to supersede or contradict definitions found elsewhere in the USP–NF.]
Apparent solubility: The empirically determined solubility of a solute in a solvent system where insufficient time is allowed
for the system to approach equilibrium or where equilibrium cannot be verified. The apparent solubility may be either higher
or lower than the equilibrium solubility due to transient supersaturation or incomplete dissolution and insufficient time to reach
equilibrium.
Aqueous solubility: Solubility in a medium that is primarily comprised of water but may also contain solubilization
enhancement from co-solvents, surfactants, complexing agents, pH, or other co-solutes. The term “aqueous solubility” is very
general and should not be confused with the water solubility. The aqueous solubility is significantly affected by the composition
of the aqueous medium.
Dissolution: The non-equilibrium process of approaching the solubility limit at thermodynamic equilibrium (i.e., the solute
and solvent forming a uniformly mixed solution). The dissolution rate will affect the time required to reach equilibrium and
may affect the apparent solubility, but will not affect the final equilibrium solubility.
Equilibrium solubility: The concentration limit, at thermodynamic equilibrium, that a solute can dissolve into a saturated
solution when excess solid is present. Equilibrium can be defined as sufficiently converged when it no longer changes
significantly during a certain time frame.
Intrinsic solubility: The solubility of the uncharged (neutral) moiety. Intrinsic solubility can only be accurately measured
in pH ranges where the distribution of species is dominated by the uncharged molecule. For some compounds, it may be
impossible to directly measure the intrinsic solubility and it must be determined by fitting the solubility data as a function of pH.
Solubility: The extent to which a solute may be uniformly dissolved into a solvent. This may be referred to as equilibrium
(saturated) solubility to differentiate it from apparent solubility. Solubility may be stated in units of concentration such as
molality, mole fraction, mole ratio, weight/volume, and weight/weight.
13
Water solubility: Solubility in pure water. Measurements of solubility in pure water are problematic due to poor control
of pH and ionic strength.
REFERENCES
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bioavailability and bioequivalence studies for immediate-release solid oral dosage forms based on a biopharmaceutics
classification system (draft guidance). Rockville, MD: Food and Drug Administration; May 2015. Biopharmaceutics,
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3. Ran Y, Yalkowsky SH. Prediction of drug solubility by the general solubility equation (GSE). J Chem Inf Comput Sci.
2001;41(8):354–357.
4. Jorgensen WL, Duffy EM. Prediction of drug solubility from structure. Adv Drug Deliv Rev. 2002;54:355–366.
5. Llinas A, Glen RC, Goodman JM. Solubility challenge: can you predict solubilities of 32 molecules using a database of
100 reliable measurements? J Chem Inf Model. 2008;48(7):1289–1303.
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7. Oliveira C, Pessoa A, Costa L. Micellar solubilization of drugs. J Pharm Pharmaceut Sci (www.cspscanada.org). 2005;8(2):
147–163.
8. Loftsson T, Jarho P, Masson M, Jarvinen T. Cyclodextrins in drug delivery. Expert Opin Drug Deliv. 2005;2:335–351.
9. Sheng JJ, Sirois PJ, Dressman JB, Amidon GL. Particle diffusional layer thickness in a USP dissolution apparatus II: a
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combined function of particle size and paddle speed. J Pharm Sci. 2008;97(11):4815–4829.
10. Verma S, Kumar S, Gokhale R, Burgess DJ. Physical stability of nanosuspensions: investigation of the role of stabilizers on
Ostwald ripening. Int J Pharm. 2011;406:146–152.
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hydrochlorothiazide as model compound. J Pharm Biomed Anal. 2008;46:335–341.
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12. Völgyi G, Baka E, Box KJ, Comer JEA, Takács-Novák K. Study of pH-dependent solubility of organic bases. Revisit of
Henderson–Hasselbalch relationship. Anal Chim Acta. 2010;673:40–46.
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potentiometric acid/base titrations and calculated solubilities. J Pharm Sci. 2005;94(1):1–16.
14. Box KJ, Völgyi G, Baka E, Stuart M, Takács-Novák K, Comer JEA. Equilibrium versus kinetic measurements of aqueous
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solubility, and the ability of compounds to supersaturate in solution—a validation study. J Pharm Sci. 2006;95(6):1298–
1307.
15. Brittain HG. Solubility methods for the characterization of new crystal forms. In: Adeyeye MC, Brittain HG, eds.
Preformulation in Solid Dosage Form Development. London: Infroma; 2008:323–346.
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18. Avdeef A, Fuguet E, Llinàs A, Ràfols C, Bosch E, Völgy G, et al. Equilibrium solubility measurement of ionizable drugs–
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Rockville, MD: USP; 2018.
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Printed on: Wed Dec 13 2023, 07:09:35 AM(EST) Status: Currently Official on 13-Dec-2023 DocId: GUID-B21E4B4E-B5EF-4EA6-879E-2D03A2476ABB_3_en-US

á1236ñ SOLUBILITY MEASUREMENTS

Bovine Simulated Ruminal Fluids

1 The terms used in this chapter are defined in the Glossary.

Thermodynamic Equilibrium and Solubility

ΔGmix = ΔHmix − TΔSmix

ΔGmix = Gibbs free energy of mixing

ΔHmix = HUU + HVV − HUV

ΔHmix = enthalpy of mixing

γUV = solute-solvent interfacial surface tension

which under dilute conditions can be simplified to:

hi = entropic effect on solvent of group i with area Ai

Methods of Estimating Aqueous Solubility

logS0 = 0.5 − 0.01(MP − 25) − logKOW

S0 = intrinsic solubility (of the unionized molecule)

EFFECT OF SALTS AND COUNTER-IONS

For the salt dissolution, AnBm(s) → nA+(aq) + mB−(aq)

EFFECT OF COMPLEXING AGENTS

EFFECT OF SURFACE AREA (DISSOLUTION RATE)

C = concentration of the solute in the solvent at time, t

D = diffusivity of the solute

EFFECT OF SURFACE ENERGY

Methods for Determination of Equilibrium Solubility

Intrinsic Determination (Rotating Disk)

Physical Assessment of Solubility

SOLUBILITY MEASUREMENTS IN BIORELEVANT MEDIA (22–26)

Human Fasted-State Simulated Gastric Fluid (FaSSGF) (24)

Sodium chloride 34.2 2.00

Sodium taurocholate 0.08 0.047

Lecithin 0.02 0.015

Human Fed-State Simulated Gastric Fluid (FeSSGF) (24)

Sodium hydroxide (q.s. to pH 5) (q.s. to pH 5)

Sodium chloride 237.0 13.85

Sodium acetate 29.75 2.441

Milk, whole 1:1 1:1

Human Fasted-State Simulated Intestinal Fluid (FaSSIF-V2) (24)

Sodium hydroxide 34.8 1.392

Sodium chloride 68.62 4.010

Sodium taurocholate 3.0 1.766

Human Fed-State Simulated Intestinal Fluid (FeSSIF-V2) (24)

Sodium hydroxide 81.65 3.226

Sodium chloride 125.5 7.460

Sodium taurocholate 10 5.89

Lecithin 2.0 1.65

Glyceryl monostearate 5.0 1.79

Sodium oleate 0.8 0.24

Human Simulated Colonic Fluid—Proximal Colon (SCoF2) (25)

Acetic acid, glacial 170 10.2

Human Simulated Colonic Fluid—Distal Colon (SCoF1) (25)

Potassium phosphate, monobasic 0.24

Sodium phosphate, dibasic 1.44

Canine Fasted-State Simulated Gastric Fluid (FaSSGFc pH 1.2–2.5) (26)

Sodium chloride 14.5 0.847

Sodium taurocholate 0.10 0.055

Sodium taurodeoxycholate 0.10 0.054

Lecithin 0.025 0.019

Lysolecithin 0.025 0.012

Sodium oleate 0.025 0.0076

Canine Fasted-State Simulated Gastric Fluid (FaSSGFc pH 2.5–6.5) (26)