Drug Development and Industrial Pharmacy

ISSN: 0363-9045 (Print) 1520-5762 (Online) Journal homepage: https://www.tandfonline.com/loi/iddi20

A review of methods for solubility determination

in biopharmaceutical drug characterisation

Ardita Veseli, Simon Žakelj & Albin Kristl

To cite this article: Ardita Veseli, Simon Žakelj & Albin Kristl (2019): A review of methods for
solubility determination in biopharmaceutical drug characterisation, Drug Development and
Industrial Pharmacy, DOI: 10.1080/03639045.2019.1665062

To link to this article: https://doi.org/10.1080/03639045.2019.1665062

Accepted author version posted online: 06

Sep 2019.

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A review of methods for solubility determination in biopharmaceutical

drug characterisation

Ardita Veseli, Simon Žakelj* and Albin Kristl

Department of Biopharmaceutics and Pharmacokinetics, University of Ljubljana, Faculty of

Pharmacy, Ljubljana, Slovenia

Correspondence: Simon Žakelj; E-mail: simon.žakelj@ffa.uni-lj.si

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A review of methods for solubility determination in biopharmaceutical

drug characterisation

Abstract

The significance of thermodynamic solubility in biopharmaceutical compound or drug

characterization as well as the importance of having methods that accurately establish it

have been extensively addressed. Nonetheless, its precise determination continues to

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remain a challenging task to accomplish. Even more so when the number of compounds

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to evaluate is high and the available amount of each compound is low, both of which

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are inevitable for the compound characterization during the drug development process.

Except for the shake-flask method which is still considered as the ʻgold standardʼ in
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obtaining thermodynamic data, it is currently difficult to say that another satisfactory

model which is routinely used to determine thermodynamic solubility is being applied.

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Therefore, this review summarizes the various experimental approaches which are
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based on the classical shake flask method but have yet attempted to speed up the

experimental process of obtaining such data more conveniently. The most important
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experimental features of these approaches are provided to the reader. Some advantages
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and disadvantages associated with each approach are also highlighted, consequently

offering a resource to those looking for the most appropriate of the approaches that
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have already fared well at determining the biopharmaceutically relevant drug solubility.

Keywords: Biopharmaceutical drug solubility; Thermodynamic (equilibrium)

solubility; Shake flask method; High throughput; Miniaturized assays

Introduction
It is well established that solubility as a physicochemical and biopharmaceutical property

has acquired an essential role in drug discovery and development. This parameter in

concurrence with permeability must be known to categorize a drug according to the

Biopharmaceutical Drug Classification System (BCS), which was first introduced by Amidon

et al. [1] and serves as a predictive approach to correlate the in vitro drug dissolution with the

in vivo bioavailability. Among all biopharmaceutically unfavourable compound properties,

poor solubility is on the top of the list. Unfortunately, poor aqueous solubility has often led to

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failure in drug development and has been manifested by the increased presence of BCS class

II and class IV compounds [2-4]. Determining the accurate and biopharmaceutically relevant

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solubility of drug substances is a challenge which should not be underestimated. In fact, due

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to the complicated solubilisation process and solid phase chemistry of drug candidates, the
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determination of solubility can be a complex task [5].

Moreover, the introduction of various terms such as ʻintrinsic solubilityʼ, ʻapparent

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solubilityʼ, and ʻkinetic solubilityʼ has subsequently stirred up some confusion in the

terminology of the field [6–10]. In this paper, our focus lies on thermodynamic solubility
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which even to this day is considered to be the more accurate representation of true solubility
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[6]. Thermodynamic solubility seeks to establish the extent to which the compound dissolves
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and it manifests the equilibrium between the solution and undissolved stable polymorph.

These values are not absolute numbers either, since the thermodynamic value depends on the
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properties of the compound and various experimental factors which involve buffer

composition, time for attaining equilibrium, temperature, mixing conditions, polymorphism,

compound purity, and particle size and shape [2,7,8].

Even though, kinetic measurements can be useful for screening in the early discovery

stage, thermodynamic solubility is still preferred in predicting drug properties and

establishing structure property relationship (SPR) during lead optimization. It is also

generally recommended to avoid the incorporation of kinetic solubility data in ADME

prediction tools and employ high quality aqueous solubility measurements instead [2]. In this

review, our goal is to characterize and provide a comparison of available methods used to

experimentally determine thermodynamic solubility. First, we start with the classical shake-

flask method and move onto assays that have tackled the issues of throughput, sample

requirement and analytical sensitivity, all of which are key factors in facilitating such

experimental determinations. Further, the strengths and limitations of these methods are

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discussed since the type of method employed is also an essential aspect in solubility

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assessment.

Shake-Flask Method

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The shake-flask method which was first introduced more than 50 years ago, is still
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considered as the most reliable technique in establishing solubility and comprises of the

following steps: (1) sample preparation, (2) equilibration, (3) separation of phases, (4)
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analysis of the saturated solution and residual solid and (5) data analysis and interpretation.
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An amount of excess solid which does not have to be accurately measured is added to the

aqueous buffers [9]. This is because in principle, solubility should not be impacted by the
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amount of solids added, as long as we are strictly working with only one species of the solid
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and with one species of the solute while the pH of the dissolution medium is constant. When

the solubility of the tested free acid or free base is reasonably low, the second condition is
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easily met by the use of buffers with sufficient capacity. The first condition, however, is often

overlooked in pharmaceutical applications simply because of the overwhelming interest in

the direct measurements of the solubility of salts at biologically relevant pH conditions.

Very few drugs are neither acids nor bases. The salts of free acids or bases are

favoured to some extent due to better technological properties but especially because of their
superior dissolution and solubility. Inevitably, the dissolution of a highly soluble salt at a pH

which favours the un-ionised form will result in precipitation of the less soluble un-ionised

form. In this case, the excess of the solid salt dictates the rate of total dissolution, but the

equilibrium solubility of the salt is never achieved since the precipitation of the free base or

acid – a competing process with its own kinetics – serves as a continuous solute sink. The

competition between the dissolution and the precipitation can result in a steady state solute

concentration which is in fact neither the solubility of the salt nor that of the free base or acid,

but somewhere in between depending on the excess of the solid salt, the medium pH and

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other experimental factors. In these very common cases, the biological relevance of the

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experimental setting including the excess solid is helpful in the biopharmaceutical

interpretation of the acquired results. Below, there is a summary of a few studies in which

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the authors have observed and attempted to explain some atypical effects of the presence of
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solid excess in the saturated solution.

Wang et al. [10] presented such a case showing that the quantity of the excess solid
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used affected the solubility of a dihydrochloride salt of a diprotic weak base. Essentially, the
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concentration of the counterion, chloride was altered in concomitance with the change in the

amount of excess solid. In a pH region 2-5 where the monohydrochloride salt dictates the
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solubility, the drug which was firstly added as a dihydrochloride salt was converted to its
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monohydrochloride salt and chloride ions were released. As a result of this release, the

solubility of the drug was supressed via the common ion effect. However, in this case, the
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physicochemical property of the drug needs to be emphasized. The compound possesses two

pKas (3.1 and 7.7) and the disparity between the two pKas is what contributed to the

alterations in solubility in the pH region 2-5 based on the added amount of solid. Therefore,

the change in solubility should be attributed to the physico-chemical property of the drug

rather than the intrinsic effect of excess solid on solubility [11].

 Contrarily, Kawakami et al. [12] investigated the effect of excess solid on the

apparent solubility of free acids and a base at various pH conditions. Out of all the studied

drugs, indomethacin was the one that showed higher dependency of solubility on the amount

of excess solid. The apparent solubility seemed to decrease with an increase in the solid

amount at pH 5 and 6 while it increased with an increase in the solid amount at pH 6.5 and 7.

The authors suggested that the impact of the solid amount on the solubility of indomethacin

seemed to be most likely affected by a competition between the crystallization and

dissolution rates. In fact, the measurement of indomethacin solubility is often associated with

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difficulties, most likely because several polymorphic solid forms with different solubility of

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this drug exist and therefore, its solubility may appear to change over time [13].

Nonetheless, there are other issues in this study that need to be addressed, such as the

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lack of buffer concentration and buffer capacity values whilst the pH values were not
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measured once the samples reached equilibrium. Murdande et al. [11] has suggested that the

decrease in indomethacin solubility at pH 5 could be derived from the suppressed ionization

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of the drug. Whereas, the increase in solubility at pH 7 might ensue from self-association or
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partial formation of sodium salt of indomethacin or even the possibility of co-crystal

formation [11]. On the other hand, according to the results obtained in the study by Baka et
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al. [7], hydrochlorothiazide solubility did not depend on the amount of solid excess in the
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solution, a finding which was not in contradiction with the results of Higuchi et al. [14]. Then

again, the authors did recommend using a small amount of excess (5-10 mg/5ml) for the
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purpose of avoiding difficulties in sampling [7].

It is generally agreed that equilibrium in shake-flask solubility determinations is

reached when the same apparent solubility of the analysed samples is given after different

equilibration time periods. Equilibrium can be frequently reached within 24 hours with good

agitation. However, it is possible to find reported stirring times from 48 h to 2 weeks [15-17].
This is the case especially when working with compounds that are poorly soluble. When the

effective surface area for dissolution is increased, the procedure can be accelerated by either

vortexing or sonicating samples prior to equilibrium evaluation. Throughout the equilibration

process, amorphous material could be added to the samples or sample temperature may be

manipulated [18]. This will help create a supersaturated solution which serves as an

alternative in solving the issue of slow dissolution rate [9]. Poor wettability and tendency to

float are additional challenges that tend to rise when establishing solubility of poorly soluble

compounds [19]. These can often be resolved by means of small glass microspheres which

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will de-aggregate the particles with agitation or sonication [9] - the two approaches that we

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also often utilize in our laboratories.

To obtain biopharmaceutically relevant drug solubility data, it is recommended to

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implement a physiologically acceptable pH range. The U.S FDA (Food and Drug
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Administration) guidelines [20] recommend a pH range of 1 – 7.5, while the EMA (European

Medicines Agency) [21] proposes to conduct solubility studies along the pH of 1.2 – 6.8 at a
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temperature of 37 ± 1 °C. More importantly, the pH should be verified and in some cases
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readjusted at the beginning of the experiment and before the separation of phases. This is

particularly crucial in the case of ionizable compounds which exhibit a pH-dependent

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solubility [22, 23] and are prone to overwhelming the buffer capacity when their solubility is
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high enough. The change of the medium pH caused by the dissolved tested compound results

in underestimated solubility for free acids and bases or in overestimated solubility for their
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respective salts. Apart from adjusting the pH with an appropriate acid or base solution it is

also worth considering the incorporation of a buffer with sufficient buffering capacity in a

repeated experiment.

During the separation of phases, filtration and centrifugation are the obvious choices.

As easy as filtration sounds, in the case of hydrophobic and poorly soluble compounds, filter
sorption may be a significant source of error. This is especially an issue when the sample

volumes are small. When filtration is inconvenient, centrifugation or ultracentrifugation are

chosen instead. The separation of the solid from the saturated solution should take place at

the equilibrium temperature, especially when equilibrium is reached rapidly. Filtration at

ambient temperature can be tolerated for poorly soluble compounds since in this instance,

equilibrium is reached slowly [9]. The saturated solutions are analysed by UV

spectrophotometric method or HPLC analysis. The use of HPLC is more frequent due to its

advantage of detecting impurities and instabilities. Lastly, several techniques allow the

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identification of any solid-state changes as well. Such techniques include powder X-ray

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diffraction (PXRD), differential scanning calorimetry (DSC), microscopy, Raman and

infrared spectroscopy, and solid-state NMR [24-27]. Examining and reporting crystal forms

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of excess drug solids is of great importance in such studies. In the event that the excess solid
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is not in the most stable crystalline form, then non-equilibrium solubility data could be

attained. Irrespective of its reliability, the shake flask method is time consuming as a single
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solubility experiment can take several days or even weeks. This restricts the usefulness of the
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method, making it unsuitable for medium to high throughput screening. It requires a large

quantity of the drug which is normally not available at early discovery phase hence limiting
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its suitability to the late discovery/early development stage. Hence, several attempts were
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made to miniaturize the classical shake-flask method, minimize the necessary solvent volume

and by doing so, to decrease the consumption of the tested compound and to somewhat
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increase the method throughput. The miniaturization inevitably requires compromises or

adaptations regarding the solid handling, sampling or separation of phases etc. as is described

in the examples below and summarized in the Table 2 but these methods still provide

solubility results comparable to those from the shake-flask method, can be used with various

solvents and with various tested compounds.

Miniaturized Shake-Flask Method

A miniaturized version of the shake-flask method was developed by Glomme et al.

[28] with the purpose of determining solubility accurately with a minimized amount of the

drug. It incorporates all steps of the equilibration method but on a smaller scale. An excess

amount of the drug was placed into the Whatman Uniprep filter chamber followed by the

addition of 2 mL of solvent volume. The chamber was closed with a plunger which consisted

of a filtration membrane and a pre-attached cap. The experiment was performed at 37 °C

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while the sample was agitated at 450 rpm for 24 h. The pH was checked after several hours

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and at the end of the experiment. Once the shaking process came to an end, the plunger was

put into the chamber which pushed the filtrate into the reservoir of the plunger. Finally, the

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HPLC with UV detection was used to determine the concentration of the compound in the
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filtrate.

The simplicity and inexpensiveness of the method are certainly considered advantageous.
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Solubility for almost every substance, including the neutral ones can be established while
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enabling the use of a vast range of media [28]. However, the use of the Whatman Uniprep

device may sometimes be conducive to drug loss especially for the poorly soluble compounds
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due to adsorption on the device and especially the filter membrane [29]. In addition, surface
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tension and volume-to-filter surface ratio could be problematic when solubility is established

on a small-scale setting [2]. The necessary amount of substance for one determination is
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dependent mainly on compound solubility. For a poorly soluble compound, <1 mg is

required. In this case, in silico calculations are recommended, but not mandatory to assess the

approximate amount needed [28,30-33]. The HPLC is considered the only high-priced

element of this assay but nowadays, it can be found in most labs.

 Nevertheless, it is necessary to proceed with caution when implementing the low-volume

approach. If this method is applied in the analysis of salts of strong acids and bases, then the

combination of the low volume with low buffer capacity may contribute to an unstable pH

value. The miniaturized shake-flask method is not suitable for high throughput measurements

since it generates data for only approximately 20 compounds per week, but it has been stated

to be precise and reliable [28]. The lack of high-throughput seems to be compensated by the

quality of the produced data seeing as this method has been repeated several times [34-40].

Small Scale Shake-Flask Method (SSF Approach)

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Another modified version of the shake-flask method can be found in the study conducted

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by Bergström et al. [41] where the solvent volume was reduced to 1000, 500, 200, 100, and

even 50 µL. No statistically considerable discrepancies were observed in solubility values in

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this volume range. Thus, it was established that volume reduction does not affect the
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precision of the method. The study was conducted at room temperature (22.5 ± 1 °C), the

samples were added in Milli-Q water and shaken at a speed of 300 rpm. Since structurally
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diverse compounds were chosen, samples were withdrawn at different time points. The
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samples of the drugs that were expected to dissolve rapidly were withdrawn after 24, 48 and

72 h whereas, for the slowly dissolving drugs the withdrawal took place after 24, 72 and 144
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h. To diminish loss of substance and sample volume, the filtration step was replaced by
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ultracentrifugation (for 15 min. at 23000 g) and the samples were analysed by high-

performance liquid chromatography (HPLC) system. For the purpose of validation, the
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obtained results were compared to the previously published solubility values from the

traditional shake-flask method. Both sets of values were in good agreement, confirming the

reliability of the SSF method.

In order to expand the SSF approach, Bergström et al. [42] studied an additional 25 bases

over a wide pH range employing a 0.15 M phosphate buffer where the pH of the drug
suspensions was adjusted with either KOH or H3PO4 solutions. The same experimental

conditions as in the former study were employed, except that all samples were withdrawn

after 24 h. The decision was made based on the previous experiment which showed that for

most of the drugs, equilibrium is attained within this time frame [41]. However, in the study

by Avdeef et al. [43], it was tentatively indicated that 24 h might have not been enough for

the practically insoluble drugs to reach equilibrium, additionally suggesting it as a topic for

further research. In both cases some of the investigated drugs were used as their

corresponding HCl salts, so the effect of the HCl salt on solubility, which we have already

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discussed in this review from the perspective of excess solid used, could be examined. It was

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claimed that no effect was observed when the drugs were studied in Milli-Q water

nonetheless, when phosphate buffer was utilized as a solvent, a minor impact on the

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solubility of the compounds was seen [41,42]. This serves as further indication that when
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phosphate buffers are incorporated in solubility studies, caution is required due to their

interaction with weak base drugs [44,45].

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Moreover, concerns about the pH control in the study by Bergström et al. [41] were
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expressed by Völgyi et al. [44] regarding two of the analysed compounds namely

promethazine and propafenone. Therefore, implementing a rigorous protocol is vital since

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even the slightest alterations may cause inconsistencies in solubility [46]. This technique was
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deemed to be suitable for the 96-well microtiter plate format which makes it possible to

establish solubility by using only microgram quantities of samples. Through this format,
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solubility determinations can also be automated allowing high throughput measurements.

Miniature Devices

A miniaturized device was set up to measure the solubility of six compounds with

diverse chemical structures in a microscale setting in the study by Chen and Venkatesh [47].

A tube closed in a continuous loop was filled with the drug slurry (≤ 0.5 mg) in an aqueous
solution. Tubes with two different diameters, 0.8 mm and 1.6 mm were used and the slurry

was continuously pumped through the tube loop. The solid material was trapped by a filter

(0.45 µm pore) and once the solution samples were removed, they were injected into the

HPLC for quantitation. The method was validated by comparing the results to the ones

obtained by shake-flask assay. When a limited amount of drug is available, which is normally

the case in early drug discovery, it was stated that a quantity as low as 0.2 mg can be added to

the smaller tubing to determine solubility.

In the traditional shake-flask method, drug adsorption to the filter membrane might

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pose a significant obstacle. Hence, a benefit of this method is the minimization of adsorption

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due to the drug slurry being constantly filtered through the membrane [46,47]. However, due

to the continuous pumping of the drug slurry, filter blockage could be an issue [48].

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Equilibrium was reached within 6 h of circulation for most compounds which is seen as an
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advantage as it facilitates a shorter turnaround time. This can be quite beneficial in building

structure-solubility relationships. With this set-up only 5-10 compounds are tested per roller
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pump however, the possibility of setting up the device for high throughput measurements
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exists according to the authors. Bearing in mind the challenging process of pumping highly

viscous excipients or semi-solid and solid excipients, this technique is mostly restricted to
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aqueous or low viscosity pharmaceutical vehicles [46,47]. Additionally, the use of syringe
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filters may impose difficulties in terms of solid sample recovery. With most of these filters it

is not possible to reclaim the solid, which in turn hinders the process of determining the
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crystalline form of the solid phase that would influence drug solubility.

Equilibrium 96-well Microtiter Plate Methods

Several studies have reported to have measured thermodynamic solubility by

implementing the solvent evaporation method. Firstly, the compound is dissolved in an

organic solvent, which is later removed by evaporation. This step is followed by the addition
of the aqueous medium to the drug for the purpose of incubation. Once equilibrium is

attained, separation is achieved usually by centrifugation and ultimately solubility is

determined by HPLC [48]. The solvent evaporation methods were developed to tackle

practicality issues such as excessive weighing tasks or automation of the solid dispensing

process of small drug quantities. However, the pre-dissolution of the drugs in organic

solvents, particularly DMSO followed by evaporation has caused somewhat of a concern.

This is due to the limited ability of some of these protocols to distinguish the polymorphous

states of the reconstituted solid samples. Moreover, some of these methods seem to have

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found their use primarily in in-house solubility measurements as it has also been stated by the

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authors themselves [49-51].

The Thermodynamic Solubility Assay (THESA Method) through which it is possible

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to establish solubility in the lead optimization phase in some of the key solvents has been
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reported by Alsenz and Kansy [2]. An aqueous buffer solution is added to a glass tube

containing the sample which is afterwards ultrasonicated for 1 h, shaken for 2 h and allowed
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to stand for another 20 h. No information is provided regarding the temperature control which
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would be crucial especially during the 1h long sonication which could cause overheating of

the sample. Once the pH of the saturated solution is determined, the solution is either filtered
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or centrifuged and finally, the concentration is determined by ultra-fast HPLC or LC-MS.

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The Partially Automated Solubility Screening (PASS Method) which was also

described by Alsenz et al. [52] was developed for selecting vehicles for preclinical studies
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while parallelly allowing thermodynamic solubility measurements in aqueous, non-aqueous,

high and low viscosity solvents in early pre-formulation. Typically, a compound (100 mg) is

suspended or dissolved in heptane to achieve a final concentration of 25 mg/mL.

Subsequently, 20-160 μL of the drug solution or suspension is pipetted into 96-well plates

and the vehicle is removed by means of evaporation in a SpeedVac centrifuge [52]. Very
much like with the “solvent evaporation method”, this approach in relation to the issue of

solid handling also results in a risk of the formation of new physical forms. Concerns have

also been raised due to the dispersion of the crystalline compound in heptane. An accurate

amount of compound in solid suspension can be difficult to dispense, especially when dealing

with low volumes [48]. Therefore, to ensure that the original physical form was maintained

even after vehicle removal, differential scanning calorimetry (DSC) and thermogravimetric

analysis (TGA) were performed. After evaporation, 40-80 μL of solvent and stir bars were

added to each well which were capped, and the plates were stirred at 300 rpm for 24 h at

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room temperature. Centrifugation was executed twice. The first time (for 1 min. at 2000 g)

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and the second time (for 15 min. at 3200 g), to remove any remaining solid from the

supernatant. The samples were eventually quantified by UHPLC. Combined with a robotic

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liquid-handling system, this technique has a throughput of 45 samples per hour and therefore,
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>600 solubility measurements can be obtained per week [52].

Arguments as to why this method is useful were provided. Based on the generated
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results, an example of how digoxin doses may be formulated was given. It is possible to
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assess the feasibility of a dosage form intended to be given to animals based on the solubility

in a given solvent, solvent toxicity and maximum dose volumes per route. Estimations of the
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in vitro and in vivo behavior of compounds and formulations can be made based on the
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solubility results. This technique may also serve as a tool for selecting the suitable excipients

to improve the dissolution rates or bioavailability of low solubility drugs. Nonetheless, the
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inability to analyze changes in the residual solid is the shortcoming of this assay. As a result,

in later stages or when more compound is available, PASS assay is usually replaced by the

SORESOS (Solubility and Residual Solid Screening) assay. Also, when screening viscous,

semi-solid and solid excipients, the PASS method might not be convenient due to the
challenging dispensing process of these excipients into a 96-well plate while using a robotic

liquid-handling system [48].

Wyttenbach et al. [53] established SORESOS (Solubility and Residual Solid

Screening), a combined screening method for solubility measurement and residual solid

screening in aqueous and non-aqueous solvents. This combination provides a new outlook for

the formulators in choosing vehicles for preclinical studies. The assay required 100 µL of

solvent, 10-15 mg of compound and single-use stirring bars per well in a 96-well microtiter

plate. The plate was sealed, and the drug-vehicle slurries were mixed by head-over-head

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rotation at 20 rpm for 24 hours at room temperature. The liquid and residual solid were

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separated by centrifugation at 3,300 g for 5 min and the drug concentration was determined

by UHPLC whereas the residual solid drug was assessed by high-throughput (HT)

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transmission X-ray Powder Diffraction (XRPD). When assessing residual solid, XPRD is
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considered as gold standard however, when low amounts of sample are available Raman

microscopy might serve as an alternative as well [53].

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Caffeine, carbamazepine and piroxicam were chosen as model compounds for

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analysis. The results correlated well to the traditional shake-flask method and to other

published data. It is an easy assay to perform and according to the amount of data that it
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generates, it is classified as medium throughput. Still, the authors concluded that it is suited
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for high throughput experimentation and automation. Due to the parallel measurement of

solubility and of crystal form, it is applicable in drug development and pre-formulation.

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Problems arise when screening high viscosity excipients and therefore, it is limited to

screening of the low viscosity ones [48,53].

Potentiometric approaches

Avdeef [54] was the one who initially introduced potentiometric based approaches

into solubility profiling however, two methods which are relatively demanding from the
perspective of necessary equipment are available at present, the pSol Gemini and CheqSol

systems (Table 1) [2]. For these two methods which run on a semiautomatic basis, the pH

electrode and capillary dispenser tips are placed in the glass tube which is necessary for the

process of titration. After the pH electrode is calibrated and the titration solutions are

standardized, an acid-base blank titration is performed which is needed to calculate solubility

and measure the electrode error. For the pSol method an octanol water partition coefficient

(logP) is required to calculate solubility by using the Hansch equation whereas and an exact

pKa/pKb is fundamental to determine solubility [28,55].

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With the pSol method, the time to determine solubility depends on the compound

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which sometimes might be more than 24 hours. The same can be said concerning the

substance requirements as the amount depends principally on compound solubility. In silico

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calculations to estimate the amount required are obligatory for pSol and an initial weight of
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<100 μg is considered sufficient. This amount has been reported to be adequate for a pH

profile determination as well. Additionally, the workload in the laboratory when using pSol is
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lighter. It is viewed as an economical method in terms of compound consumption and the

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Food and Drug Administration (FDA) considers it acceptable for the evaluation of solubility

for BCS classification [56]. Nevertheless, it is limited to ionizable compounds only. The
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pH/solubility profiles determined by pSol method have also been referred to as the industry
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ʻgold standardʼ [57]. The potentiometric nature of the method also means that the medium

options are limited. Lastly, the method is a low throughput one as it can generate solubility
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data for only about 3 poorly soluble compounds per week [28].

As for CheqSol (Chasing Equilibrium), it was first introduced by Stuart et al [58]. It

has been reported that the technique assesses kinetic, thermodynamic (equilibrium) and

intrinsic solubility. Additional advantages were highlighted when excipients were tested in

terms of drug formulation in a study by Fornells at al. [59] since the method permits the
determination of both, the extent and duration of supersaturation. This is predominantly

significant for poorly soluble drugs as supersaturation is often employed as a strategy to

improve their bioavailability. CheqSol contains five stages which include dissolution, seeking

precipitation, additional precipitation, chasing equilibrium and re-dissolution. Prior

knowledge of the pKa of the compound is necessary. Initially, the kinetic solubility is

established from the concentration of the solution when a precipitate first appears as the pH

of a solution of the drug in ionized form is adjusted by adding acid or base titrant to convert

the substance to its unionized form.

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As the experiment continues, the equilibrium solubility is subsequently determined in

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the presence of precipitated neutral solid, by monitoring pH changes induced by precipitation

and actively seeking equilibrium pH where equal amount of the sample is precipitating and

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dissolving per unit time. The quantity of the tested solid that is needed for establishing
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solubility must ensure a concentration above its intrinsic solubility when fully neutral to

allow precipitation once the pH is adjusted accordingly. If substance precipitation is not

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detected, a larger quantity is required. A minimum of 2-5 mg was reported for the
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experimental volume of 10 mL, but for substances that exhibit a higher solubility, larger

amounts are of course necessary [58]. Only 20 - 80 minutes are required for kinetic and
pt

equilibrium solubility determination per sample, making it faster than the pSol method [60].
ce

Alas, the method is also restricted only to ionizable compounds. Seeing as the CheqSol

method essentially considers the Henderson-Hasselbalch equation always valid since

Ac

pH/solubility profiles cannot be directly measured, in some cases it could potentially lead to a

source of systematic error [44]. Nevertheless, the error would not affect the experimentally

established kinetic or intrinsic values but only the pH/solubility profiles.

Column Elution Method

As indicated by the name, the apparatus employed in the column elution method

consists of a microcolumn that contains either glass beads, silica gel or sand all of which are

inert carrier material, together with an excess of test substance. A small plug of glass wool is

used to keep this inert material in place. Constant temperature in the column needs to be

sustained. The preferred eluent is double distilled water however, deionized water with a

resistivity above 10 megohms/cm and a total organic carbon content below 0.01% can be

used. When connected to a recirculating pump, the headspace of the column should have the

t
ip
capacity for at least five bed-volumes of water and five samples. In order to remove water

cr
soluble impurities, the first five bed volumes are discarded. The recirculating pump can be

either a peristaltic or membrane pump by which a flow of approximately 25 mL/h

us
(corresponding to 10 bed volumes per hour) should be efficiently maintained. At this point, it
an
is necessary to be cautious as contamination and/or adsorption may occur with the tube

material. Alternatively, the microcolumn can be connected to a levelling vessel in which a

M

flow rate of approximately 25 mL/h is required too [61,62].

ed

Once the eluate fractions are collected, the next step requires to check if colloidal

matter is present by means of the Tyndall effect. If any particles are present, it indicates a
pt

poor filtering action of the column, rendering the test invalid. Gas or liquid chromatography,
ce

titration, photometric or polarographic methods are appropriate for sample analysis.

Equilibrium is attained when the mass concentration of the eluate is constant. In contrast to
Ac

the shake flask method (Table 1), the column elution method applies to substances with low

solubilities. It is not applicable to volatile substances and in order to determine solubility,

substances must be as pure and stable in water. Nevertheless, further research is still vital in

finding the most suitable carrier material, as the ones previously mentioned may not yet be
optimal. The construction of the column and regulation of the flow rate require optimization

as well [61,62].

Conclusion

Due to the status of solubility in the design of drug compounds as well as in the

development and optimization of drug manufacturing processes, an accurate understanding

and determination of this property is valuable in drug discovery. While medium to high

throughput assays certainly have their advantages, low throughput solubility measurements

are still performed to provide more accurate information relevant to the formulation strategies

t
ip
and overall profile of the drug candidate developability [63]. The idea of having only one

cr
consistent solubility method for both, the discovery and development phase may seem

desirable at first however, various factors such as compound quantity, speed and workflow

us
differences prevent such a set-up [2]. Even today, solubility assays are not ʻone size fits allʼ
an
therefore, different methods are applied at different stages of drug discovery and

development [8]. Thus, recognizing the limitations of each assay is vital so the data could be
M

interpreted properly.
ed

Acknowledgement
pt

This research was financially supported by Slovenian Research Agency (ARRS) [P1-0189].
ce

Disclosure statement
Ac

No potential conflict of interest was reported by the authors.

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Table 1. Comparison between shake flask, potentiometric and column elution methods
Shake Flask Method Potentiometric Methods Column Elution Method
(SF) (pSol, CheqSol)
 Simple experimental  Lighter workload  Simple experimental
set-up  Lower compound set-up
 Inexpensive consumption in  Inexpensive apparatus
 Short time to reach
ADVANTAGES

apparatus comparison to SF
 Easy to learn  Useful in drug saturation compared to SF
 Most accurate formulation studies (e.g.
method to date in investigating
excipients)
 Established solubility
values correlate well with
SF

t
 Time consuming  More demanding in  Large drug quantity is

ip
 Labour intensive terms of laboratory required
 Not suitable for high equipment  Better suited carrier

cr
throughput  Limited medium options material is desirable
DISADVANTAGES

screening  Low throughput  Loading of carrier material

 Large drug quantity

us
 Restricted to ionizable can be problematic when
is required compounds the substance is deposited
as an oil or different
an
crystal phase (inaccurate
results may be generated)
 Optimization of column
M

construction and flow rate

are still desired
ed
pt
ce
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Table 2. Comparison of miniaturized methods based on the classical shake flask (SF) method
Miniaturize
96-well Plate
d Shake Small Scale Shake Miniature
Methods (PASS,
Flask Flask Method Devices
SORESOS)
Method [41,42] [47]
[48-53]
[28]
“medium” “medium”, but
“low” with “high”: 600
approx. 20 96-well format and
Throughput possibility to measurements / week
compounds/ automation are
increase for the PASS method
week possible

Fast
Solid dispensing Slow Slow Slow
(solvent evaporation)

Experiment 6h for most 23h (THESA)

t
24h 24h or more
duration compounds 24h (SORESOS)

ip
cr
Filtration
after
Continuous

us
equilibration
Ultra- filtration,
Phase separation , Centrifugation
centrifugation filter blockage
filter
isues
sorption
an
possible

Necessary demanding –
simple, More demanding
M

equipment besides simple complex

consumable equipment in most cases
(U)HPLC device
ed

Solid sample
Yes, especially for
recovery and Difficult Possible Difficult
SORESOS method
analysis
pt

Risk of formation High (PASS), but was

of new solid Low Low Low controlled by DSC and
ce

physical forms TGA

Aplicability in
High, especially PASS
Ac

studies of Possible Possible Low

method
excipient effects