Comparison of Nephelometric, UV-Spectroscopic, and HPLC Methods

Mar 24, 2009 - Woldeamanuel A. Birru , Dallas B. Warren , Sifei Han , Hassan Benameur , Christopher J. H. Porter , Colin W. Pouton , and David K. Chal...
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Anal. Chem. 2009, 81, 3165–3172

Comparison of Nephelometric, UV-Spectroscopic, and HPLC Methods for High-Throughput Determination of Aqueous Drug Solubility in Microtiter Plates Bettina Hoelke,† Sabine Gieringer,† Michael Arlt,‡ and Christoph Saal*,† Merck KGaA, Frankfurter Strasse 250, 64293 Darmstadt, Germany, and EMD Serono Research Institute, One Technology Place, Rockland, Massachusetts 02370 High-throughput screening (HTS) is an important method in the pharmaceutical industry for discovering hits that will be further developed into leads, clinical candidates, and eventually into medicines. After an HTS campaign, solubility studies of the discovered hits are an important means to judge the validity of the pharmacological result and to prioritize them for further studies. In the present paper, different methods for determination of kinetic solubility were compared that are able to provide sufficient throughput and that consume only small amounts of compound. In particular nephelometric determination, UV-spectroscopic determination, and determination of kinetic solubility by HPLC have been investigated. These three assays have been compared with regard to their detection limit, information content, and speed/throughput. Further on, parameters influencing solubility in the specific HTS assay which may vary for different HTS assays are discussed. These include formation of different salt forms, time used for incubation during the assay, and concentration of cosolvent. Finally a comparison of the results and purpose of kinetic and thermodynamic solubility is given. Among the physicochemical parameters which are important in drug discovery and development, solubility plays a key role.1 During lead optimization and pharmaceutical development, solubility is investigated in depth because of its influence on drug bioavailability.2-4 Hence, it is desirable to have high solubility when entering lead optimization. High-throughput screening delivers often hundreds or thousands of hits from a single screening campaign. During the process of hit evaluation or during the hit to lead process, one is often faced with the fact that a number of those hits constitute * To whom correspondence should be addressed. E-mail: christoph.saal@ merck.de. Fax: +49 6151 723073. Phone: +49 6151 727634. † Merck KGaA. ‡ EMD Serono Research Institute. (1) Lipinski, C. J. Pharmacol. Toxicol. Methods 2000, 44, 235–249. (2) van de Waterbeemd H.; Lennerna¨s H.; Artursson P. Drug Bioavailability; Wiley-VCH: Weinheim, Germany, 2004. (3) Curatolo, W. Pharm. Sci. Technol. Today 1998, 1, 387–393. (4) Avdeef, A. Curr. Top. Med. Chem. 2001, 1, 277–351. 10.1021/ac9000089 CCC: $40.75  2009 American Chemical Society Published on Web 03/24/2009

false positives.5 Limited solubility of compounds can be one origin of unspecific effects as it may lead to precipitation or aggregation of compounds.6 This scenario defines a set of requirements for methods used to determine compound solubility in a hit to lead process. It is quite different from the requirements for experiments used to evaluate solubility during lead optimization and drug development with the goal of guaranteeing appropriate solubility to optimize drug bioavailability. The hurdles for the techniques used to investigate solubility for HTS are in some respects higher than for evaluating solubility to optimize bioavailability. In some respects they are even simpler to handle: (1) As regards throughput, the number of compounds which require the determination of solubility to warrant a suitable starting point for HTS is magnitudes larger than the number of solubility determinations during lead optimization and drug development. For beneficial use in hit evaluation, it becomes crucial to access the solubility of at least a larger fraction of hits from HTS. This may amount to thousands of compounds per year. The throughput of the method used for solubility determination must be comparable to the throughput of methods which are used in parallel for the qualification of HTS compounds, e.g., check of identity and purity.7,8 (2) For accuracy, solubility determinations relevant for assessing bioavailability and optimization of research compounds during the lead optimization phase require quantitative values with a high accuracy and precision in a thermodynamic sense. By contrast, the goal after HTS is to make sure that precipitation does not occur on the time scale of the HTS assay. As a consequence, methods used at this stage are helpful even if they just will give roughly the range of solubility. This becomes especially obvious if one bears in mind that the aqueous systems used in HTS runs usually vary from one assay to the next. These different assay conditions will also influence the solubility to a certain extent. (3) With material demand, a further requirement originates from the amount of substance which is available during lead finding. Usually, screening compounds are available only in milligram (5) Seidler, J.; McGovern, S. L.; Thompson, N.; Shoichet, B. K. J. Med. Chem. 2003, 46, 4477–4486. (6) Kerns, E. H. J. Pharm. Sci. 2001, 90, 1838–1858. (7) Yurek, D. A.; Branch, D. L.; Kuo, M. S. J. Comb. Chem. 2002, 4, 134–148. (8) Yan, B.; Fang, L.; Irving, M.; Zhang, S.; Boldi, A. M.; Woolard, F.; Johnson, C. R.; Kshirsagar, T.; Figliozzi, G. M.; Krueger, C. A.; Collins, N. J. Comb. Chem. 2003, 5, 547–559.

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quantities, whereas during later stages of research and development the amount of substance becomes less critical. Because substances usually used for HTS are dissolved in dimethylsulfoxide (DMSO) at a concentration of 10 mM, it becomes beneficial if these DMSO stocks can also be used for the determination of solubility. (4) The use of DMSO stock solutions also becomes crucial in terms of the respective kinetic conditions. Whereas solubility determinations starting with solid material represent dissolution experiments, those determinations using DMSO stock solutions represent precipitation experiments. (5) For automation, a further practical argument for the use of DMSO stock solutions comes from the stringent requirement of automated methods for solubility determination. Using solid materials would make automation more complicated because solid state handling, e.g., by powder pipets usually is much more demanding than dosing of DMSO stock solutions. Liquid pipetting can be done by robots using microtiter plates as a standard format. Nevertheless, other methods for introduction of compounds into assays for determination of solubility are described in the literature.9 In contrast to the assays for determination of kinetic solubility in the HTS environment, as described in the present work, these methods are more focused to the early development phase. Altogether these requirements set the stage for the methods used to evaluate solubilities prior to or directly after HTS. Certainly the easiest and fastest methods to assess solubility for several hundred thousands of compounds are in silico tools giving the respective information very quickly.10 A further benefit comes from the fact that in silico methods can be used even in the design of HTS libraries, before the synthesis of the respective compounds. Nevertheless, in silico methods might have drawbacks in that they usually do not take sufficiently into account the state of ionization at a certain pH. In addition, they might be conservative as far as they only determine the solubility in a pure aqueous system. In practice, certain components present in the assay, e.g., DMSO, may act as cosolvents leading to better prevention of precipitation. Taken together, this discussion leads to the conclusion that a fast and fully automated method for the determination of aqueous solubility using 10 mM DMSO stock solutions, or similar concentrations, will be helpful for the qualification of HTS compound pools or hits if the purpose of the method is discussed carefully. In regards to analytical methods for high-throughput determination of kinetic solubility, these have been described in the literature 11-15 and to a lesser extent for thermodynamic methods.14,15 However, there is no discussion in the literature taking into account the effect of solid state forms in the determination of kinetic solubility and comparing these results to thermodynamic solubility. In the above-mentioned literature, assays for the determination of kinetic solubility starting from DMSO solutions by nephelometry, UV-spectroscopy, and HPLC are described and compared. Alsenz, J.; Meister, E.; Haenel, E. J. Pharm. Sci. 2007, 96, 1748–1762. Taskinen, J. Curr. Opin. Drug Discovery Dev. 2003, 3, 102–107. Bevan, C. D.; Lloyd, R. S. Anal. Chem. 2000, 72, 1781–1787. Quatermain, P.; Bonham, N. M.; Irwin, A. K. Eur. Pharm. Rev. 1998, 18, 27–32. (13) Pan, L.; Ho, Q.; Tsutsui, K.; Takahashi, L. J. Pharm. Sci. 2001, 90, 521– 529. (14) Chen, T.-M.; Shen, H.; Zhu, C. Comb. Chem. High Throughput Screening 2002, 5, 575–581. (15) Roy, D.; Ducher, F.; Laumain, A.; Legendre, J. Y. Drug Dev. Ind. Pharm. 2001, 27, 107–109. (9) (10) (11) (12)

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Quatermain et al.12 present a method for ranking of solubility. Pan et al.13 compare kinetic solubilities as obtained by nephelometry, UV-spectroscopy, and HPLC. However, this comparison is only semiquantitative as many of the test compounds exhibit solubility >5 × 10-4 mol/L, and solubility higher than this concentration is not differentiated further but taken into account for calculation of correlation coefficients. Roy et al.15 briefly describe two protocols for measurement of thermodynamic solubility by UV-spectroscopy. The first protocol starts from solid material. The second protocol introduces the compounds as acetonitrile solution, and acetonitrile is evaporated without control of the solid state form obtained by the evaporation procedure. Chen et al.14 compare assays using UV-spectroscopy and HPLC for determination of kinetic and thermodynamic solubility. However, none of the authors address the difference in the solid state form which is in equilibrium with the solution of the compound. Generally, it is expected that different solid state forms might be encountered during determination of kinetic and thermodynamic solubility. In assays for determination of thermodynamic solubility, the compounds are introduced as crystalline material or might recrystallize during incubation with buffer. In contrast, for determination of kinetic solubility, the compounds are introduced into the aqueous solvents predissolved in organic solvents, mainly DMSO, and precipitate very fast. This can lead to amorphous precipitates. In this work, beyond comparison of different analytical methods for assessment of solubility, we point out the different natures of kinetic and thermodynamic solubility originating from the different solid state forms and discuss the different purpose of the methods as a consequence of this behavior. EXPERIMENTAL SECTION Materials. Test Compounds. The compounds investigated in this study were either research compounds or commercial compounds selected to represent a wide variety of solute properties. These properties include solubility, lipophilicity and acidic/ basic behavior. Commercial compounds were purchased as follows: glybenclamide, loxapine-succinat, zolpidem, beclomethasone-dipropionate, albendazole (Sigma Aldrich); tolnaftate, fludrocortisone-acetate, 8-methoxypsoralen, 2-(4-thiazolyl)-benzimidazol, flunarizine-dihydrochloride, econazole, miconazole, tamoxifen, fenofibrate (Prestwick); loperamide-hydrochloride (ABCR); betamethasone-21-acetate (TCI Deutschland); sitosterin (VWR International); terbinafin (Specs). Compounds were dissolved in DMSO (Merck KGaA, Uvasol, part no. 102950) to yield 10 mM stock solutions. Buffer Medium. To 2.383 g of HEPES, (2-[4-(2-hydroxyethyl)1-piperazinyl]-ethanesulfonic acid, Merck KGaA, part no. 110110) 7 mL of 1 N sodium hydroxide (Merck KGaA, part no. 109137) was added and diluted by Millipore water to yield a volume of 500 mL. The buffer solutions were filtered by sterile filters (0.22 µm, Corning Inc., part no. 430769) and checked for pH which was 7.40 ± 0.05. 96-Well Plates Used for Nephelometric Assay. For the nephelometric assay, only one kind of microtiter plates was required. Preparation of the sequential dilution row as well as measurement of turbidity was carried out on the same type of 96-well microtiter plates (Thermo Electron, part no. 9502227).

96-Well Plates Used for UV-Spectroscopic and HPLC Assay. For UV-spectroscopic measurements, 96-well plates (Greiner Bio-One GmbH, part no. 655801) were used. For these plates, a typical UV-absorption of 0.1 absorption units was determined at 230 nm. The filtration step was carried out using the MultiScreen vacuum filtration system (Millipore part no. MAVM 096 OR). This set consisted of the respective 96-well filter plates and a tub which was placed below the filter plate and held another 96-well microtiter plate collecting the filtrates. The tub and the filter plate were hermetically connected by a seal allowing application of vacuum to accelerate the filtration step. After the filtration step, acetonitrile (Merck KGaA, part no. 100016) was added to the filtrates to prevent precipitation of the dissolved compounds. Instrumentation. Kinetic Solubility: Nephelometric Assay. The laser nephelometer used in this study was the Nephelostar (BMG LabTechnologies). The setup of this instrument consisted of a red diode laser (632.8 nm) located vertically above an automated mount for 96-well plates and an integration sphere. The integration sphere was positioned below the 96-well plates. The detector was placed in a 90° position. A black absorber in the 180° position made sure that only light scattered from the suspension reached the detector. The signal measured by the detector increased linearly with particle concentration for up to 3 orders of magnitude. Saturation effects were observed at very high particle concentrations. Formazin standards as described by the European Pharmacopeia16 were used to check the working range and linearity of the nephelometer. Special attention was paid to the microtiter plates used for the nephelometric measurements because rugged or scratched surfaces of the plates can lead to a significantly increased lower limit of detection of turbidity. All nephelometric measurements were carried out with an integration time of 0.5 s. This setup yielded a time of approximately 2 min for scanning one 96-well mictrotiter plate. Kinetic Solubility: UV-Spectroscopic Assay. Measurement of UV spectra from samples in 96-well plates was carried out on a SpectraMax Plus plate reader (Molecular Devices, Sunnyvale). This plate reader yielded access to the spectral range 190-1100 nm. However, because of the absorption from the 96-well plates and the nature of the chromophors present in the test compounds spectra were only taken in the range 230-400 nm. The spectral bandwidth was set to 2 nm. The data point interval was chosen to be 10 nm. The empty plate holder was used as the reference channel. The reference and sample were measured sequentially. A check of the photometric linearity of the UV plate reader using solutions of K2Cr2O7 at variable concentrations led to a linear range of the instrument from 0.003-3.5 absorption units for wavelengths 257, 313, and 350 nm.17 Kinetic Solubility: HPLC Assay. A Merck Hitachi L4200 HPLC equipped with a diode array detector and a LiChroCart 125-4 Lichrospher 100 RP-18 column (Merck KGaA, part no. 150943) was used. Eluent A consisted of 2 mL of diethylamine (Merck KGaA, part no. 803010) in 1000 mL of methanol (Merck KGaA, part no. 106018), eluent B of 5 g of ammonium acetate (Merck KGaA, part no. 101115), 5 mL of methanol and 995 mL of water. A flow rate of 1.0 mL/min was used. During 15 min, the ratio of (16) European Pharmacopoeia, 6th ed. (6.2); Council of Europe: Strasbourg, France, 2008; Chapter 2.2.1: Nephelometry. (17) European Pharmacopoeia, 6th ed. (6.2); Council of Europe: Strasbourg, France, 2008; Chapter 2.2.25: Absorption Spectroscopy.

eluent A/eluent B was changed from 25:75 to 95:5. From 15 to 19 min, the ratio of eluent A/eluent B was kept constant at 95:5. Thermodynamic Solubility: HPLC Assay. An Agilent 1100 HPLC equipped with a diode array detector and a Chromolith RP18e column (Merck KGaA, part no. 152001) was used. Eluent A consisted of 0.1% formic acid (Merck KGaA, part no. 100264) in water, eluent B consisted of 0.1% formic acid in acetonitrile (Merck KGaA, part no. 100016). A flow rate of 0.85 mL/min was used for t < 5.5 min and 2.5 mL/min for t g 5.5 min. For t < 0.6 min eluent A/eluent B was kept constant at 90:10. Until 4 min, this ratio was changed to 10:90 and kept constant until 5.5 min. Powder X-ray Diffraction. XRD diffactrograms were collected with a Bruker AXS D5000 in transmission mode with a primary monochromator. Samples were exposed to Cu KR-1 radiation (40 kV, 30 mA, 1.540 56 Å). The instrument was operated in the step scan mode, in increments of 0.05° 2θ over 3-65° 2θ, and the counts were accumulated for 1.4 s at each step. Samples were prepared between two Mylar foils. Description of Assays. Kinetic Solubility: Nephelometric Assay. The starting point to determine solubilities by the nephelometric assay consisted of preparing a row of suspensions with different total concentrations, sum of dissolved and undissolved substance, covering the range from 5 × 10-7 to 5 × 10-4 mol/L. This set of suspensions was prepared directly in the 96-well plate used to obtain the nephelometric values on the plate reader. The higher concentrations of the dilution row were obtained by mixing different volumes of buffer and 10 mM DMSO stock solution. For concentrations lower than 10-4 mol/L, the suspensions were prepared by sequential dilution taking an aliquot from the well with the next higher concentration which was prepared in the previous step and diluting this. For all suspensions, a concentration of DMSO of 5% was realized by adding the respective amount of pure DMSO to the wells. The final volume of all suspensions was set to 300 µL. Kinetic Solubility: UV-Spectroscopic Assay. The UV-spectroscopic assay required a standard plate used for calibration of absorption as a function of concentration of the respective research compound. A second plate contained the saturated solution which was obtained by the filtration step. The calibration plate contained solutions of the research compounds in concentrations ranging from 7.6 × 10-9 to 5 × 10-4 mol/L. The preparation of the dilution row was carried out by pipetting defined volumes of 10 mM DMSO stock solutions into the wells and adding a mixture of 80% of the buffer and 20% acetonitrile. The acetonitrile acts as a cosolvent to prevent precipitation. This procedure was used for concentrations above 10-4 mol/L. For preparation of solutions with lower concentrations, a similar dilution procedure was applied as described for the nephelometric assay. For all solutions of the dilution row, the content of DMSO was fixed to 5% by adding the respective amounts of DMSO into the wells. The final volume in each well was 285 µL. In each dilution row, one well without research compound was added. The UV spectrum of this blank was used for offset correction. For measurement of the UV spectra, 200 µL of the respective solution was transferred to a UV-transparent plate. To obtain saturated solutions of the research compounds, 10 µL of 10 mM DMSO stock solution was added to 190 µL of buffer. Analytical Chemistry, Vol. 81, No. 8, April 15, 2009

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Figure 2. Calibration of UV-spectroscopic measurement for solubility of compound A by the UV-spectroscopic assay, a 3-fold repetition of the assay on different well plates. Figure 1. Determination of solubility of compound A by the nephelometric assay, a 3-fold repetition of the assay on different well plates.

Mixing these volumes was carried out directly in the filter plate. The filter plate was shaken automatically. Using these volumes of buffer and DMSO stock solutions yielded the same DMSO concentration as present in the calibration samples described above. After 90 min, the suspensions were filtered into another 96-well plate. From this 96-well plate, 160 µL of each solution was transferred to the 96-well microtiter plate used for taking UV spectra. Before these spectra were measured, 40 µL of acetonitrile was added to prevent any further precipitation. Kinetic Solubility: HPLC Assay. For calibration of the HPLC assay as well as for the preparation of saturated solutions of the research compounds, the same procedures were applied as described for the UV assay. The transfer steps to the 96-well plates used for measurement of the UV spectra were omitted. Thermodynamic Solubility: HPLC Assay. Thermodynamic solubility was determined by the shake flask method. About 2 mg of the respective compound was weighed into a Whatmann UniPrep Syringeless (part no. UN113UORG) Filter and shaken for 24 h with the buffer. Amounts of solid materials were chosen to make sure that a solid residue remained during the incubation. After 24 h, the suspensions were filtered by pushing down the plunger of the syringeless filters and the concentration of the compounds was determined by HPLC with UV detection. For quantification, reference solutions of the compounds in a mixture of acetonitrile and methanol (1:1) were used. RESULTS AND DISCUSSION Kinetic Solubility: Nephelometric Assay. Obviously, the nephelometric assay is the most straightforward approach to get access to solubilities by high-throughput techniques. This is due to the fact that this approach starting from 10 mM DMSO stock solutions only requires pipetting and no handling of solids or a filtration step. In addition, no transfer step is required as the setup of the test solution and the determination are carried out on the same plate. The technique was described previously by Bevan11 and Pan.13 Beyond the results described there, some limitations of the technique have to be discussed. Figure 1 shows the turbidity of compound A as a function of concentration. The assay was carried out 3 times independently using different 96-well plates. The reproducibility of the assay was extremely high leading to a 3168

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solubility of 8 × 10-5 mol/L. However, if compounds with solubility below 2 × 10-5 mol/L were investigated this yielded very similar dependencies of the measured turbidity on sample concentration within the dilution row. In such cases, an intercept of the linear increasing turbidity signal with the constant offset obtained for low concentrations at 2 × 10-5 mol/L was observed. This result was due to the fact that the concentration of the suspended compound in the respective wells of the dilution row was so low that the amount of precipitate did not lead to light scattering with intensity above the background signal. Accordingly a lower detection limit of about 2 × 10-5 mol/L has to be stated for the nephelometric assay as described here. In addition, this lower detection limit depends significantly on the background signal obtained from blank solutions and the quality of the well plates used in the assay. Kinetic Solubility: UV-Spectroscopic Assay. For the UVspectroscopic assay, a filtration step was necessary. However, in comparison with the HPLC assay which requires the filtration step too, the analytic step was much faster. The measurement of an UV spectrum in the range 250-400 nm took about 5 s. Extending this spectral region toward shorter or longer wavelength did not lead to further benefits. Below 250 nm, DMSO has a strong UV absorption. There are only very few research compounds with chromophors absorbing light above 400 nm. Most compounds in HTS pools possess between one and three aromatic or heteroaromatic ring systems leading to significant UV absorptions in the range 260-300 nm which is most suitable for evaluation of aqueous solubility. A typical calibration curve as obtained from a dilution row with eight concentrations is shown in Figure 2. A suitable concentration range for the assay has been found to comprise concentrations from 5 × 10-7 to 5 × 10-4 mol/L. The optical path length for UV measurement in a 96-well microtiter plate using a sample volume of about 300 µL is in the range of 4 mm. Considering this optical path length and typical lower and upper limits for absorption coefficients of HTS compounds of 500 and 20 000 L/(mol cm), respectively, this yields absorption values ranging from 0.0001 (εmin ) 500 L/(mol cm), c ) 5 × 10-7 mol/L) to 4 (εmax ) 20 000 L/(mol cm), c ) 5 × 10-4 mol/L). Correlation coefficients obtained from repeated UV measurements with eight different concentrations typically were found in the range 0.99 to 0.9999 indicating good linearity

Figure 3. Calibration of HPLC measurement for solubility of compound A by the HPLC assay, a 3-fold repetition of the assay on different well plates.

and reproducibility. For practical application of the assay, the high correlation coefficient allowed the use of one or two-point calibrations. This reduced the number of time-consuming dilution steps during preparation of the calibration set. Furthermore, the linearity observed in the calibration curve clearly showed that a good degree of reproducibility of the optical path length has been reached. In principle, this parameter could be critical since the optical path length in well plates is not set to a fixed value as in typical UV cuvettes but determined by the meniscus of the liquid. The latter depends mainly on the surface tension of the solution. It can be concluded that the surface tension is determined to a major degree by the buffer system and the DMSO but not by the different concentration of the research compounds under investigation. Despite the high precision of the UV assay, a drawback might be incomplete information about adsorption of compounds on the filter plates which might lead to systematic error. The same holds true for the HPLC method using the identical filtration step. Kinetic Solubility: HPLC Assay. The wet chemical part of the HPLC assay is identical to the UV-spectroscopic assay with regard to sample preparation up to the filtration step. However in comparison with the UV-spectroscopic assay, the nephelometric assay is much slower. In our case, the HPLC assay requires about 19 min for running one chromatogram. The calibration curve obtained from a dilution row with 10 concentrations ranging from 8 × 10-9 to 5 × 10-4 mol/L is shown in Figure 3. The lower concentration gives the limit of detection of the HPLC method. This limit of detection is obtained by comparing the HPLC signal with the 3-fold noise level. As for the UV method, reproducibility and linearity are good leading to correlation coefficients higher than 0.999. Consequently, also for this assay the calibration can be performed as a one or two-point method. The detection wavelength has been set to 254 nm even though the HPLC method circumvents DMSO absorption allowing the use of shorter wavelength for detection. Comparison of Nephelometric Assay, UV-Spectroscopic Assay, and HPLC Assay for Determination of Kinetic Solubility. Results obtained from kinetic solubility determinations by the three different methods are shown in Figure 4a for 29 research compounds and in Figure 4b for 18 commercial compounds. For solubility above the limit of detection of the nephelometric method, a good correlation of the results obtained from all three assays

was found. As one moves toward the limit of detection of the nephelometric method, the solubilities obtained by this method approach the limit of 2 × 10-5 mol/L as described above. For solubilities above the detection limit of the UV-spectroscopic method, there is good agreement between the UV-spectroscopic and the HPLC assay. For lower concentrations, the HPLC assay offers the only access to solubilities in this range. With comparisons of the minimum solubility which can be determined by the three assays (>2 × 10-5 mol/L for the nephelometric method, >5 × 10-7 mol/L for the UV-spectroscopic method, and >8 × 10-9 mol/L for the HPLC method), the HPLC method clearly is the most sensitive. Nevertheless, for practical use in many cases, the UV-spectroscopic assay will yield sufficient information to evaluate if poor solubility can compromise results of high-throughput screening assays because typical HTS test concentrations are 10-6 mol/L or higher. Limitations which prohibit the use of the UV-spectroscopic assay and HPLC assay can result as follows: (a) Compounds without chromophors cannot be detected by the UV-spectroscopic and by the HPLC assay. However, this will not represent a serious drawback for these methods because typically the large majority of compounds in HTS pools will possess at least one aromatic or heteroaromatic system. (b) For two of the research compounds, sticky precipitates which could not be filtered were obtained from the addition of DMSO stock solution to the aqueous buffer system. This behavior can also be problematic in the nephelometric assay because for these compounds it is difficult to obtain good mixing of the aqueous suspensions which is crucial for the following dilution step. Even if the nephelometric assay in these cases yields a result, in an automated setup a failing filtration step may not be detected easily. An error in subsequent dilution steps in the nephelometric setup will be difficult to recognize. (c) Impurities in the research compounds which can contribute to increased solubility cannot be discriminated in the nephelometric and the UV-spectroscopic assays. However, they will be found easily in the HPLC assay. Whether this represents a critical issue or not depends very much on the quality of the compounds. If purity of the compounds has been checked at an earlier stage or in parallel, e.g., by HPLC-MS, HPLC-UV, HPLC-ELSD, or a combination of these methods,6,7 generally, a second investigation of purity will not be necessary. If purity or stability of the compounds in buffer is unknown, attention should be paid to this when investigating the aqueous solubility. Kinetic Solubility: Influence of Incubation Conditions. So far we have discussed the different analytical methods used in the three different assays for solubility determination. Beyond method-inherent limitations of the methods, the operations used to generate a precipitate will have an influence on the results of solubility determinations, too. In the following section we will discuss parameters such as the kind of buffer system, incubation time, and concentration of DMSO. These parameters may affect precipitation of different salts and polymorphs. Time-dependent conversion of these forms can lead to different kinetic solubility profiles. Another parameter which can lead to artifacts, especially for compounds exhibiting low solubility, is adsorption of compounds on filter membranes. This effect is described elsewhere in the literature11 and will not be further mentioned here. Analytical Chemistry, Vol. 81, No. 8, April 15, 2009

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Figure 4. (a) Comparison of results of kinetic solubility determinations by the nephelometric (4), UV-spectroscopic (0), and HPLC (O) assays using 29 research compounds. The compounds have been arranged according to decreasing solubility by HPLC assay. (b) Comparison of the results of solubility determinations by the nephelometric(4), UV-spectroscopic (0), and HPLC (O) assays using 18 commercial compounds. Additionally these results are compared with results from thermodynamic solubility measurements by HPLC (b).

Buffer Systems. In HTS, a variety of buffer systems is used. For research compounds exhibiting basic or acidic properties which lead to the presence of ionic species at the respective pH value of the assay, formation of different salts with the respective anions and cations of the buffer system becomes possible. The solubility of the salts formed in different buffer systems may vary significantly for one compound. Different solubilities of pharmaceutical salts have been discussed by Stahl.18 When discussing solubility of research compounds, one has to distinguish between thermodynamic and kinetic solubility. Kinetic aspects of solubility are important in a clinical setting where in vivo conditions are simulated by dissolution testing.19,20 In an HTS environment, another aspect of kinetic solubility is more important as solubility is approached by precipitation. This may lead to different kinetic solubilities of a given compound depending on whether the experiment is carried out using solid or predissolved compound. Incubation Time. The solid state form, e.g., whether a precipitate is crystalline or amorphous, has an influence on the measured solubility. The effect of polymorphic forms and amorphous forms on solubility is discussed by Hilfiker21 and Pudipeddi et al.22 and will be further treated in the next section by comparing kinetic and thermodynamic solubility. For the kinetic solubility assays discussed in this work, these solid state properties come into play by the following mechanism: After addition of the DMSO stock solution to the aqueous buffer system, precipitation takes place. This might be a very fast process or proceed even more slowly depending on the ability of a certain compound to undergo spontaneous nucleation, the speed of crystal growth, and the ability to form oversaturated solutions in the specific buffer-DMSO (18) Stahl, P. H.; Wermuth, C. G. Handbook of Pharmaceutical Salts. Properties, Selection, and Use; Wiley-VCH: Weinheim, Germany, 2002. (19) Kaus, L. C.; Gillespie, W. R.; Hussain, A. S.; Amidon, G. L. Pharm. Res. 1999, 16, 272–280. (20) Vertzoni, M.; Dressman, J.; Butler, J.; Hempenstall, J.; Reppas, C. Eur. J. Pharm. Biopharm 2005, 60, 413–417. (21) Hilfiker R. Polymorphism in the Pharmaceutical Industry; Wiley-VCH: Weinheim, Germany, 2006. (22) Pudipeddi, M.; Serajuddin, A. T. M. J. Pharm. Sci. 2005, 94, 929–939.

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Figure 5. Solubilities of research compounds determined after different incubation times determined by HPLC assay.

system. To investigate this effect, two research compounds were incubated in different experiments using incubation times ranging from 10 min to 24 h. The results are displayed in Figure 5. For both compounds it can be clearly seen that longer incubation times lead to lower solubility. For the two compounds investigated, we found relative solubility differences of a factor 5-10 when comparing values at 10 min versus 24 h. This effect can be mainly attributed to the crystallinity of the precipitates. The conditions used in HTS assays, fast addition of DMSO stock solutions to the aqueous buffer system, usually prohibit formation of crystalline materials. Instead, in many cases amorphous precipitates are obtained. This has been confirmed by X-ray powder diffraction. Results are shown in Figure 6. These amorphous materials exhibit higher solubility than crystalline materials. If incubation is prolonged, recrystallization takes place leading to lower solubility. Concentration of DMSO. To investigate the effect of DMSO concentration on the solubilities, two compounds were chosen. The HPLC assay was carried out using concentrations of DMSO of 0.5 vol %, 1 vol %, 2 vol %, and 5 vol %. To vary the DMSO concentration, different concentrations of the compounds in DMSO stock solutions, which were prepared especially for this purpose, were used. To obtain the solubility in the pure buffer

CONCLUSION

Figure 6. Powder X-ray diffractograms of a research compound as crystalline material (lower trace) and precipitate obtained from addition of 10 mM DMSO stock solution to the buffer system and a consequent filtration step (upper trace). The upper trace has been shifted vertically to improve representation.

Figure 7. Solubilities of research compounds determined in systems with different DMSO concentrations by HPLC assay.

system, without any DMSO, the solubility of both compounds was determined by the shake flask method using the compounds as solid material. It can be seen from Figure 7 that variation of DMSO concentration from 0.5 vol % to 5 vol % leads to an increase in solubility by a factor of approximately 5 for compound a and approximately 2 for compound b. The solubilities of the solid materials are slightly lower than those obtained for systems with 0.5 vol % DMSO, pointing out that for these specific compounds, precipitation of amorphous material is not an issue. Thermodynamic Solubility: HPLC Assay. As discussed above, methods for the determination of the kinetic solubility in many cases yield solubility referring to amorphous materials. From this, it is expected that solubility obtained from kinetic methods generally yields results which are higher than solubility as determined by thermodynamic methods. Comparison of kinetic solubility determined by nephelometric, UV-spectroscopic, and HPLC assay versus thermodynamic solubility is depicted in Figure 4b. It can be clearly seen that for the vast majority of the compounds, solubility measured by the thermodynamic assay is considerably lower than solubility as obtained from the kinetic methods. This result clearly reflects the effect of the morphic form of the solid residue in equilibrium with the solution.

In this study we compared three methods which can be used for fast determination of kinetic solubility of compounds in HTS libraries. Nephelometric determination of solubility has the advantage of avoiding a filtration step slowing down the turnover rate of the assay. Precipitation of compounds dissolved in DMSO stock solutions, incubation, filtration, and subsequent determination by UV spectroscopy still provides a fast method for solubility determination. As HPLC detection is used for determination of analyte concentration in the filtrate, the analytical method becomes the bottleneck in the assay. However, in terms of the limit of quantitation, the inverse order is obtained: Determination by the nephelometric assay can be carried out for solubilities higher than 2 × 10-5 mol/L. The UV-spectroscopic assay including a filtration step yields access to solubilities down to 5 × 10-7 mol/L and finally substituting the UV-spectroscopic determination by the HPLC method renders determination of solubilities down to 8 × 10-9 mol/L possible. With the use of these values to consider which method is most suitable for checking solubilities of compounds in HTS pools, it has to be kept in mind which concentrations of the respective compounds are most frequently used in biological assays to determine primary assay results, IC50 and EC50 values. In many cases these parameters are determined using conditions where the highest concentrations involved in the respective assay, and consequently the highest risk for precipitation exists, are in the range of 10-5 mol/L. Accordingly, only in rare cases will there be the need to use HPLC detection in solubility determination, and the nephelometric assay or UV-spectroscopic assay will gain sufficient information. Furthermore, it has to be kept in mind that there are more parameters influencing precipitation in biological HTS assays. These include the nature of the buffer system which might cause improved solubility due to the presence of cosolvents or might deliver lower solubility, for example, by formation of salts exhibiting decreased solubility. The presence of proteins in cellular assays can also have an influence on solubility. HTS assays are usually carried out by adding DMSO stock solutions to aqueous buffer systems. For most compounds this represents very harsh conditions for crystallization leading to amorphous precipitates in many cases. In terms of solubility, this improves the assay since oversaturated solutions become accessible. As a consequence, calculated solubilities can be regarded as a worst case scenario since in silico methods generally refer to thermodynamic solubilities for crystalline materials. Second, calculated solubilities can be considered as a worst case scenario because they refer to aqueous systems ignoring the effect of DMSO acting as a cosolvent. Nevertheless, this effect should not be overemphasized since, even for DMSO concentrations as high as 5%, this effect produces an increase in solubility which is below 1 order of magnitude. Finally, all methods discussed can be automated on robotic systems where 96-well plates are used. In terms of speed, cost, and maintenance, clearly the nephelometric assay is superior to the assays using a filtration step. However, in terms of the amount of information obtained, the ranking of the three methods becomes reversed. Analytical Chemistry, Vol. 81, No. 8, April 15, 2009

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Through comparison of kinetic and thermodynamic solubility from this study, it becomes clear that kinetic solubility in many cases refers to amorphous solid residues whereas thermodynamic solubility mainly refers to crystalline material. Accordingly, solubility obtained from kinetic measurements generally leads to results reflecting higher solubility. With regard to the intended use of this information, this factor has to be taken carefully into account: Whereas kinetic solubility clearly reflects a situation close to that found in many high-throughput screens and does answer the question of whether a compound precipitates in the highthroughput screen, it is not a useful tool for optimization of compounds as regards to solubility. Clearly, for optimization of solubility, thermodynamic solubility has to be taken into account, preferably including information on the solid state form. Using

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kinetic solubility for optimization of a research compound bears the risk of not optimizing the compound into the direction of higher solubility but into the direction that makes it harder to crystallize. ACKNOWLEDGMENT We gratefully acknowledge Joachim Maerz, Mirek Jurzak, and Johannes Dasenbrock (Merck KGaA) for fruitful discussion and in silico prediction of physical chemical parameters.

Received for review January 2, 2009. Accepted March 5, 2009. AC9000089