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1 Research Article ISSN: Lakshmi Narasimham Y S et al. / Journal of Pharmacy Research 2011,4(2), Available online through Kinetic and intrinsic solubility determination of some b-blockers and antidiabetics by potentiometry Lakshmi Narasimham Y S 1* and Vasant D Barhate 1 1 Department of Chemistry, Vivekanand Education Society s College of Arts, Science and Commerce, Chembur, Mumbai , India Received on: ; Revised on: ; Accepted on: ABSTRACT In this paper the ph-equilibrium solubility profiles of some beta-blockers and anti-diabetic compounds are presented. The equilibrium solubility values of glibenclamide, glipizide, gliclazide, glimepiride, propranolol, atenolol and amlodipine besylate were determined using the saturation shake-flask and the Chasing Equilibrium Solubility (CheqSol) methods. Results obtained by the two methods are in good agreement. In this procedure, the equilibrium solubility is actively sought by changing the concentration of the neutral form by adding HCl or KOH titrants and monitoring the rate of change of ph due to precipitation or dissolution. In the case of glibenclamide and glipizide, due to limited solubility of its protonated forms at high ph, same was determined using methanol-water mixtures. Experiments were performed in varying ratios of low ionic strength methanol water mixture and results extrapolated to aqueous conditions were in good agreement with that of literature values. An extrapolated ph vs. solubility profile is then generated by calculation using the measured pka and the intrinsic solubility for all the selected drugs. Key words: Potentiometry; equilibrium solubility; intrinsic; shaker flask, HPLC 1. INTRODUCTION Solubility may be defined as the amount of a substance that dissolves in a given volume of solvent at a specified temperature. More specifically, compound solubility can be defined as unbuffered, buffered, and intrinsic solubility. Unbuffered solubility, usually in water, means solubility of a saturated solution of the compound at the final ph of the solution [1]. The traditional approach is to use a saturation shake-flask method: drug is added to a standard buffer solution until saturation occurs, followed by shaking for 24 h-7 days, removal of excess undissolved solid, and analysis of the solution by HPLC with UV or MS detection [2, 3]. In recent years, the method has been modified to work with robotic liquid-handling systems using 96-well plates [4]. Solid is removed by filtration or centrifugation before analysis. The traditional shake-flask method provides thermodynamic solubility values and is often used as a standard method against which other methods can be validated. An alternative method for thermodynamic measurements of the intrinsic solubility of the neutral form of ionizable compounds is the potentiometric acid-base titration method described by Avdeef [5]. The intrinsic solubility is the equilibrium solubility of the free acid or base form of an ionizable compound at a ph where it is fully unionized [6]. Kinetic-based methods have been introduced to meet the needs of drug discovery teams. These methods use solutions prepared from DMSO stocks and attempt to rank or classify molecules to see whether they meet some appropriate cut off criteria. The detection system is usually based on light-scattering methods using laser light or single-wavelength emissions, which detect precipitated particles in solution. The most common methods are based on the work of Lipinski [7] or use robotics and 96-well plate nephelometers [8]. Recently, a light-scattering method using flow cytometry has also been published [9]. Equilibrium solubility (the concentration of compound in a saturated solution when excess solid is present, and solution and solid are at equilibrium) can be measured by the classical shake-flask method, and while this method is accurate when performed to a high standard, it is slow. Kinetic solubility (the solubility at the time when an induced precipitate first appears in a solution) can also be measured, and while the measurements are much faster than shake-flask, the kinetic solubilities obtained are often much higher than measured equilibrium solubilities. Most drugs are ionizable in aqueous solution because they contain at least one acidic or basic functional group. These molecules can exist in neutral (uncharged) or ionized (charged) form depending on the ph of the solution. They are more soluble in charged form and their aqueous solubility is ph-dependent. When the molecule exists only in the monomer state, its ph-dependent equilibrium solubility is described by the Henderson- Hasselbalch (HH) equation [10 12]. In the case of monoprotic bases, the relationship between solubility and ph is the following: where S is the equilibrium (thermodynamic) solubility at a particular ph, S 0 is the intrinsic solubility and pka is the negative logarithm of the ionization constant of the molecule. The HH relationship can be used to predict the ph-dependent aqueous solubility of drugs when the pka and logs 0 values of the compound are known. It is frequently used in order to convert the intrinsic solubility value into the equilibrium solubility at a physiologically relevant ph [13]. *Corresponding author. Lakshmi Narasimham Y S Department of Chemistry, Vivekanand Education Society s College of Arts, Science and Commerce, Chembur, Mumbai , India Fig1:(a) Propranolol hydrochloride (PRO); (b) Atenol (ATN); (c) amlodipine besylate (AB); (d) gliclazide (GLC); (e) Glipizide (GLZ); (f) Glibenclamide (GLB) and (g) glimepiride (GLM). In this study, the kinetic and equilibrium solubilities of glibenclamide, glipizide, gliclazide, glimepiride, propranolol, atenolol and amlodipine besylate drugs were measured by chasing equilibrium, a new potentiometric titration method for measuring the equilibrium solubility of ionizable compounds [14]. The measured results of these equilibrium aqueous solubilities were then compared against conventional saturation shake-flask method [15]. Since the correct calculation of HH relationship needs precise starting values, we accurately determined the pka values [16] and measured the Log S 0 values. The main objective of our present study was to understand the co solvent effect on the solubility of drug compounds in one hand and on the other hand, provide solubility-ph profiling of all the selected drug compounds. The intrinsic solubilities measured in the presence of methanol-water mixtures for glibenclamide and glipizide was extrapolated to aqueous solubility which was in good agreement with that of literature values. The extensive literature survey, to the best of our knowledge, revealed that the data generated by this procedure for the selected drug compounds were never been reported.

2 2. EXPERIMENTAL Lakshmi Narasimham Y S et al. / Journal of Pharmacy Research 2011,4(2), 2.1 Instrumentation The apparatus used to perform the solubility determinations was a Sirius T3 titrator. SiriusT3 uses less than 0.5mg of sample for most experiments, and solubility determination now requires 1-2 mg instead of mg, used to require in GLpKa instrument. The instrument comprises three hardware modules. The unit on the left is the dispenser module, which houses the precision micro dispensers for adding water, solvents and acid/ base titrants from the reagent bottles. Also contained within this module is the UV/Vis spectrometer and light source, which is connected to a fibre optic dip probe. To the right of the dispensers, the titrator module features a moving arm with the assay probes attached ph electrode, UV dip probe, stirrer, temperature sensor and capillaries for reagent addition. There is a row of buffer and wash positions used for calibrating and cleaning the probes. This includes a flowing water wash which cleans the probes with fresh water after each experiment. At the front of the titrator is the sample position, which is temperature controlled with a peltier device (fully controlled by computer), and an additional turbidity sensing device. The unit on the right hand side is the autoloader, which has a worktable with four 48- position vial trays. It has a robotic arm which automatically picks up and moves vials to the sample position. At the front of the autoloader is an ultrasonic bath, which can be automatically used to aid the dissolution of poorly soluble compounds. All titrations were performed in 0.15 M KCl solution under nitrogen atmosphere, at 25 C, using standardized 0.5M HCl and 0.5M KOH solutions. The ph electrode was calibrated titrimetrically in the ph range Methods Reagents The following drugs viz; propranolol hydrochloride (PRO), atenolol (ATN), amlodipine besylate (AB) belonging to beta-blockers and gliclazide (GLC), glipizide (GLZ), glimepiride (GLM) and glibenclamide (GLB) belonging to anti-diabetic drugs were selected for the present investigation [Fig 1]. PRO, ATN, AB, and GLM were obtained from IPCA Laboratories Limited, Mumbai, while AML, GLC and GLZ were obtained as gift samples from Bal Pharma Limited, Bangalore. GLB was obtained from Sigma. All the drug compounds used were of pharmaceutical grade. Of the chemicals used for solubility determination, methanol is of HPLC grade from Merck. Solutions and solvent mixtures were made up of distilled water obtained from Millipore, Milli-Q (Bedford, MA, USA) purification system. Readymade 0.5 M solutions of potassium hydroxide and hydrochloric acid were obtained from Merck. Potassium hydroxide is standardized against primary standard using potassium hydrogen phthalate. Potassium hydrogen phthalate is purchased from Sigma. Di-potassium hydrogen phosphate and potassium chloride were of analytical grade from sigma Preparation of methanol-water mixture For solubility determination of selected drugs in the presence of co-solvent, a 80% v/v methanol in 0.15 M ionic strength adjusted water is prepared and used throughout our investigation Determination of the Equilibrium Solubility by Saturation Shake-Flask Method Knowing the pka values and class (acid or base) of the compounds examined, the ph of the aqueous solution was selected to assure the presence of the non ionized form in proportions to be generally higher than 99%. A low ph (0.5, 2.0, or 2.5) was selected for acids, while a high ph (11.5) was selected for bases. An excess of sample was added to 2 ml of aqueous buffers and the resulting suspension was shaken at a temperature of 37 ±0.5 C for 24h on a rotary shaker to reach equilibrium solubility. Saturated solutions were then centrifuged to decant the filtrate into HPLC vials. 1 µl of sample solution is injected and the concentration is quantified against the standard. A Waters Acquity UPLC system (Waters, USA) equipped with binary gradient pump, auto sampler, column oven and photodiode array detector (PDA) was employed for analysis. Chromatographic data was acquired using Empower 2 software. A validated method along with gradient conditions is followed as described elsewhere [17, 18]. BEH C18 UPLC column, 50mm 2.1 mm, 1.7µm is used throughout the analysis. The detector wavelength was set at 220 nm. A five point standard calibration curve containing 2, 10, 25, 50 and 100 µg/ml of each compound selected in the present study was used as a calibration mixture for quantitation. neutral species had precipitated. The occurrence of precipitation was detected using the spectroscopic dip probe, and the kinetic solubility was derived from the ph at the moment of precipitation. Additional aliquots of titrant were added to produce additional solid, after which the rate of change of ph (ph-gradient) was measured once it had settled to a sustained response. In the case of glibenclamide and glipizide, it was not possible to dissolve the protonated form fully at high ph due to its limited solubility. However, the samples were found to dissolve in water methanol mixtures. All the experimental conditions were kept constant as that of aqueous solubility except that with glibenclamide, 30% and 40% methanol was used as a co solvent at the initial stage in order to dissolve the sample at high ph in ionised form. While, in case of glipizide, methanol concentration in the range of 20% to 40% was used to dissolve the compound in ionised form at higher ph. The intrinsic solubility, determined at 0%, 30% and 40% in case of glibenclamide and 0%, 20%, 30% and 40% in case of glipizide, was then extrapolated to aqueous solubility which was compared against the literature values. In order to calculate the HH relationship for these selected compounds, the apparent ionisation constant (pska) at respective co-solvent (methanol) concentration were used to determine the equilibrium solubility Kinetic solubility A kinetic solubility value can also be determined from the data. Unlike the intrinsic solubility, which is an equilibrium value, kinetic solubility values are strongly time dependent and this dependence cannot be related in a single concentration value. These kinetic solubility values could be used as additional information relating to the degree of supersaturation that occurred but would not be expected to be reproducible between different kinetic methods. The kinetic solubility values reported here are equivalent to the concentration of the neutral species at the point in the titration where precipitation is first detected. This value gives an indication of how much of the solution remains supersaturated shortly after precipitation begins. In CheqSol experiments, precipitation is induced by first dissolving the compound in ionized form in aqueous solution such as 0.15M KCl (or water-solvent mixture), and then adjusting the ph by adding small increments of strong acid or base titrant until the unionized form of the compound begins to precipitate from solution. After measuring kinetic solubility, CheqSol goes on to measure equilibrium (or thermodynamic) solubility, which is the concentration of compound in a solution when excess solid is present, and solution and solid are at equilibrium. Once precipitation is detected, the rate of ph change with time is closely monitored. The action of a compound precipitating from a supersaturated solution or dissolving into a subsaturated solution causes reproducible gradients of ph change. These gradients are monitored, whilst small amounts of strong acid (0.5 M HCl) and strong base (0.5M KOH) are added alternately to cause the sample to fluctuate between a supersaturated and subsaturated state. By careful monitoring of these rates of ph change, the equilibrium conditions can be determined and hence an intrinsic solubility can be calculated. Intrinsic solubility (S 0 ) is the equilibrium solubility of the free acid or base form of an ionizable compound at a ph where it is fully unionized [19]. 3. RESULTS AND DISCUSSION 3.1 Bjerrum analysis A useful visualization can be obtained from the Bjerrum function (or difference curve or formation curve), which shows the average number of bound protons (hydrogen ion binding capacity) versus ph [20]. Such a graph clearly reveals values of pkas for the portions of the curve where the sample remained fully in solution and may be useful diagnostically in correction for such factors as substance purity, acidity error or carbonate content Determination of the Equilibrium Solubility by the Chasing Equilibrium Method The thermodynamic solubility of the non ionized form of the samples was determined by the Chasing Equilibrium method [14]. In this method, a quantity of substance was accurately weighed into the titration vessel and a measured volume (1.5 ml) of 0.15 M KCl solution was added. The titrations were carried out at constant ionic strength and temperature (t = 25.0±0.5 0 C) under nitrogen atmosphere. A minimum of three parallel measurements were carried out. For the samples GLB, GLZ, GLC and GLM (which were acids), a measured volume of base titrant was added to adjust the solution to a ph at which the solute was fully dissolved in its ionized form. For the samples PRO, ATN and AB (which were bases), a measured volume of acid titrant was added to achieve full dissolution. The solution of ionized solute was back titrated by adding measured aliquots of acid or base titrant until the solution became cloudy, which indicated that the poorly soluble Fig 2: Bjerrum curve for glimepiride - Acid

3 Lakshmi Narasimham Y S et al. / Journal of Pharmacy Research 2011,4(2), The Bjerrum curve for glimepiride as shown in Fig 2 shows that the experimental data fit well to the theoretical Bjerrum curve for fully dissolved glimepiride. The sample precipitated out at around ph 7.4, after which the Bjerrum curve no longer matched this theoretical pka graph. Had the sample remained in solution, the curve would be expected to follow the theoretical pka and cross the half-bound proton intercept at the pka of Fig. 3 and 4 represents the Bjerrum curves of selected compounds. Of all the measured compounds in the present study, GLM had super saturation ratios around 138, while for GLB, GLZ and GLC, the supersaturation ratio was found to be around 36, 18 and 15. This means the kinetic solubility is significantly higher than intrinsic solubility and these compounds are chasers as the neutral species forms a supersaturated aqueous solution. It precipitates slowly and would take a long time for all the substance to precipitate at a given ph. Since the supersaturation ratio of PRO and ATN was found to be greater than 1.7, these compounds also belong to chasers. However, in the case of amlodipine besylate, a primary amine a base with pka of 9.2 at 25 C and 0.15 M KCl, all the points collected during CheqSol titration, including the kinetic solubility, fall on the precipitation Bjerrum graph [Fig 5]. The supersaturation ratio for this compound is found to be 0.48 which indicates that the kinetic solubility is lesser than intrinsic solubility. Means it does not form a supersaturated solution and hence is a nonchaser. Compound like this crash out solution and reach equilibrium very quickly. Fig 3: Bjerrum curves of selected compounds - Acids Fig 5: Bjerrum curve of amlodipine besylate - Base Fig 4: Bjerrum curve of propranolol and atenolol - Bases Once the sample has precipitated, the chasing equilibrium procedure quickly brings the solution close to equilibrium with the precipitate and then oscillates between supersaturation and subsaturation with very small changes to the neutral species concentration. The data points collected during this period should all lie close to a Bjerrum function that can be easily calculated from the known data and the intrinsic solubility result, by assuming that the entire solid is in the form of the neutral species. 3.2 Chasers and non-chasers The CheqSol method of measuring solubility produces two results kinetic solubility and intrinsic solubility. It was observed from the measured data that the kinetic solubility of most of the compounds measured in this study was higher than their intrinsic solubility, except in the case of amlodipine besylate, indicating that they were in supersaturated solution at the time that precipitation first appeared. Dividing the kinetic solubility by the intrinsic solubility produces a supersaturation ratio. The experimental data are summarized in Table 1. Table 1: Measured pka, log P, solubility and Supersaturation ratio Sample pka log P Intrinsic Kinetic Supersaturation ratio solubility solubility (µg/ml) (µg/ml) Glibenclamide a Glipizide a Gliclazide Glimepiride Atenolol Propranolol Amlodipine a Extrapolated solubility Sample Sample Percentage Concn of the neutral species at zero ph gradient crossing points (µg/ml) Mean of 8 SD Literature wt (mg) of crossing solubility co solvent points (µg/ml) 2-3 GLB 0% % % GLZ 0% % % % GLC 0% GLM 0% ATN 0% PRO 0% AB 0% Table 2: Zero ph gradient crossing points for the intrinsic solubility of all the selected drugs In this study, all the selected compounds are either monoprotic acids or bases. The intrinsic solubility assays determined for all the selected compounds are used here to

4 Lakshmi Narasimham Y S et al. / Journal of Pharmacy Research 2011,4(2), Table 3: Equilibrium solubility data of the investigated compounds - Acids Glibenclamide Glipizide (Acid) Gliclazide (Acid) Glimepiride (Acid) pka = 5.38 pka = 5.16 pka = 5.54 pka = 5.62 = g/100 ml = g/100 ml = at ph 7.4 at ph 7.4 g/100 ml at ph 7.4 = g/100 ml at ph 7.4 CheqSol data CheqSol data CheqSol data CheqSol data ph S [g/100 ml] ph S [g/100 ml] ph S [g/100 ml] ph S [g/100 ml] 0% 30% 40% 0% 20% 30% 40% Stomach ph Blood ph illustrate the results obtained from chasing equilibrium. The results of 3 titrations each of selected compounds, each changing the direction of the ph gradient eight times, are summarized in Table 2. Each titration of individual compounds used a different weight of sample ranging from 2 to 60 mg. The mean solubility result with a standard deviation is less than 0.2 for all the selected compounds, which is comparable to the reported literature values [21]. An example of a chasing equilibrium graph for glibenclamide is shown in Fig 6. measured by the shake-flask method at ph 7.4 is g/100ml which is in excellent agreement with the CheqSol result g/100 ml at ph 7.4. Since it was not possible to dissolve the protonated form fully at high ph due to its limited solubility, different ratios of methanol-water mixtures was employed using CheqSol method in order to dissolve the glibenclamide sample at higher ph in the ionised form at the beginning of the titration. All the experimental conditions were kept constant as that of aqueous solubility except with 30% and 40% methanol was used as a co solvent. The intrinsic solubility of different methanol-water mixture was then extrapolated to get aqueous solubility, which was linear with a regression co-efficient of [Fig 7a]. Solubility data at each concentration level of methanol-water mixture at different ph were recorded and studied. Fig 6: Near equilibrium ph gradient of 5.2 mg of glibenclamide 3.3.Equilibrium solubility CheqSol equilibrium solubility method was applied to measure the equilibrium solubility of all the seven drug compounds selected in the present study. Results are presented in Table 3 for acids and in Table 4 for bases. Though GLZ, PRO and ATN intrinsic solubility was reported earlier by Comer et al., we have chosen PRO and ATN compounds to validate our experimental conditions to reproduce the results, while GLZ was used to study the effect of co solvent (methanol-water mixture) on intrinsic solubility. In order to confirm the reliability of log S 0 values, they were also determined for their saturated solubility at physiological ph of 7.4 by conventional shaker flask method. As it is evident from the results, there was a reasonably good agreement between two different methods. The theoretical HH curves were predicted based on the precise pka values determined by potentiometric titration using GLpKa instrument. The details are interpreted for each compound separately. Table 4: Equilibrium solubility data of the investigated compounds - Bases Propranolol Atenolol (Base) Amlodipine besylate (Base) hydrochloride (Base) pka = 9.46 pka = 9.48 pka = 9.20 = = = g/100 ml at ph 7.4 g/100 ml at ph 7.4 g/100 ml at ph 7.4 CheqSol data CheqSol data CheqSol data ph S [g/100 ml] ph S [g/100 ml] ph S [g/100 ml] Blood ph Glibenclamide Glibenclamide is a monoprotic acid (pka = 5.38). The intrinsic solubility by CheqSol method for glibenclamide is found to be at 0.17 µg/ml. Its thermodynamic solubility Fig 7: Intrinsic solubility extrapolation of glibenclamide and glipizide Up to ph 4.0 there is a constant value for the solubility of the non-ionized neutral species. At ph 8.0 the sample starts to convert to ionized form. There is a 10 fold increase in the solubility thereafter for every one ph unit and at ph 12 the solubility of the compound reaches a maximum. The maximum solubility of the ph-equilibrium solubility profile is limited by the solubility product. The literature value of solubility for this compound is found to be one log unit lower than our reported value. However, Glomme et al., had reported the solubility of this compound at ph 7.0, 37 C for 24 hrs as 11.4 µm which is in good agreement with our CheqSol method [22].

5 Lakshmi Narasimham Y S et al. / Journal of Pharmacy Research 2011,4(2), Glipizide Glipizide is also a monoprotic acid. The pka value of its sulfonyl urea secondary nitrogen is The intrinsic solubility by CheqSol method for glipizide is found to be at 1.71 µg/ml. Its thermodynamic solubility measured by the shake-flask method at ph 7.4 is g/100ml which is in excellent agreement with the CheqSol result g/ 100 ml at ph 7.4. Since it was not possible to dissolve the protonated form fully at high ph due to its limited solubility, different ratios of methanol-water mixtures was employed using CheqSol method in order to dissolve the glipizide sample at higher ph in the ionised form at the beginning of the titration. All the experimental conditions were kept constant as that of aqueous solubility except with 20%, 30% and 40% methanol was used as a co solvent. The intrinsic solubility of different methanol-water mixture was then extrapolated to get aqueous solubility, which was linear with a regression co-efficient of [Fig 7b]. Up to ph 5.0 there is a constant value for the solubility of the non-ionized neutral species. At ph 8 the sample starts to convert to ionized form. There is 10 fold increase in the solubility thereafter for every one ph unit and at ph 11 the solubility of the compound reaches a maximum. Comer et al., had earlier reported the intrinsic solubility by CheqSol method as 1.40 µg/ml which is in good agreement with our reported value Gliclazide Gliclazide is also a monoprotic acid. The pka value of gliclazide is The intrinsic solubility by CheqSol method for gliclazide is found to be at µg/ml. Its thermodynamic solubility measured by the shake-flask method at ph 7.4 is g/100ml which is in good agreement with the CheqSol result of g/100 ml at ph 7.4. Up to ph 6.0 there is a constant value for the solubility of the non-ionized neutral species. At ph 7.4 the sample starts to convert to ionized form. There is a 10 fold increase thereafter for every one ph unit and at ph 10 the solubility of the compound reaches a maximum. The literature value of solubility for this compound is found to be in good agreement with that of our reported values Glimepiride Glimepiride is a monoprotic acid. The pka value of glimepiride is The intrinsic solubility by CheqSol method for this compound is found to be at 0.18 µg/ml. Its thermodynamic solubility measured by the shake-flask method at ph 7.4 is g/ 100mL which is in good agreement with the CheqSol result of g/100 ml at ph 7.4. Up to ph 7.4 there is a constant value for the solubility of the non-ionized neutral species. At ph 8.0 the sample starts to convert to ionized form. There is a 10 fold increase thereafter for every one ph unit and at ph 12 the solubility of the compound reaches a maximum. The literature value of solubility for this compound is found to differ by 2 log units Propranolol hydrochloride The hydrochloride salt of propranolol was used for the solubility investigations. Propranolol is a monoprotic base (pka = 9.53). Its intrinsic solubility obtained by the CheqSol method is 68.3 µg/ml. Its thermodynamic solubility measured by the shake-flask method at ph 7.4 is g/100ml which is in good agreement with the CheqSol result of g/100 ml at ph 7.4. Above ph 10.0 there is a constant value for the solubility of the non ionized neutral base. Below ph 8 the free propranolol base starts to convert to cationic form. At around ph 5.0 compound is found to same as that of our observed value. Though this compound was part of Comer et al., we have selected this compound in our present study as reference compound to validate the obtained results in our experimental condition using the miniatured potentiometric instrument and also provide ph dependent equilibrium solubility Atenolol Atenolol is a monoprotic base (pka = 9.48). Its intrinsic solubility obtained by the CheqSol method is 16.5 mg/ml. Its thermodynamic solubility measured by the shakeflask method at ph 7.4 is g/100ml which is in good agreement with the CheqSol result of 208 g/100 ml at ph 7.4. Above ph 11.0 there is a constant value for the solubility of the non ionized neutral base. Below ph 10.0 the free atenolol base starts to convert to cationic form. At around ph 7.4 compound is found to same as that of our observed value Amlodipine besylate The besylate salt of amlodipine was used for the solubility investigations. Amlodipine is Source of support: Nil, Conflict of interest: None Declared a monoprotic base (pka = 9.20). Its intrinsic solubility obtained by the CheqSol method is 529 µg/ml. Its thermodynamic solubility measured by the shake-flask method at ph 7.4 is g/100ml which is in good agreement with the CheqSol result of g/100 ml at ph 7.4. Above ph 10.0 there is a constant value for the solubility of the non ionized neutral base. Below ph 8 the free amlodipine base starts to convert to cationic form. At around ph 6.0 compound is found to differ by 2 log units. 4. CONCLUSIONS In this work the ph-equilibrium solubility profiles of few beta-blockers and antidiabetic drugs were studied in order to investigate the validity of the HH relationship. The intrinsic solubility (logs0) values were independently measured by the Chasing Equilibrium Solubility method. Results are in good agreement with that literature and shaker flask method. CheqSol method is much faster than shake flask, with complete experiments lasting less than 1 hour. The new method provides equilibrium solubility values in about an hour per sample. Kinetic solubility can be measured in the same experiment, and the ratio of kinetic to intrinsic solubility gives an indication of a compound s ability to form supersaturated solutions. When a drug substance failed to dissolve in its ionized form at high or low ph, the same experiment can be repeated using methanol-water mixtures as a co solvent so that still CheqSol can be adopted in the discovery laboratory. However, there is one limitation which we found while analyzing discovery compounds is that CheqSol cannot be employed for those compounds which do not undergo ionization. This limitation can be easily overcome by shaker flask method. 5. ACKNOWLEDGEMENTS We thank Dr. John comer of Sirius for providing necessary technical input while carrying out the solubility measurement. 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