OBJECTIVE: To determine the solubility of given drug by Nogami & Shake-flask method at given pH (phosphate buffer 6.8).

REQUIREMENTS: Reagents: Drug sample, Sodium hydroxide pellets, Potassium dihydrogen orthophosphate Apparatus : volumetric flasks, beakers, measuring cylinder,spatula,pipettes

THEORY:

Solubility of a substance may be defined as maximum amount of substance which can be dissolved in a particular solvent at given temperature, pressure and condition. Solubility of a substance depends upon temperature, pressure and composition of solvent system. More specifically, compound solubility can be defined as un-buffered, buffered, and intrinsic solubility.Solubility can be measured as either a kinetic or thermodynamic value.

Thermodynamic solubility: It is performed using the solid crystalline form of a compound and is defined as the maximum amount of compound in solution once equilibrium has been reached. Such studies are typically performed during the latter stages of drug discovery or early development and may help to design formulation strategies for future in vivo studies in animals and man. The methods to determine thermodynamic solubility are:

1. Scaled down shake-flask method and 2. Solvent evaporation method.

Kinetic solubility: The measurement of the kinetic (non-equilibrium) solubility usually starts from the dissolved compound and represents the maximum solubility of the fastest precipitating material, which is typically not determined. The precipitating material can be any of the existing different solid forms of the compound, for example, polymorphic changes can happen even within minutes . Also the formation of different salts may be possible depending on the presence of ionic species in the medium. The kinetic solubility is defined to be the concentration at which the precipitation starts. Kinetic solubility measurements are designed to determine if compounds possess sufficient solubility to produce reliable data in future biological and ADME testing. As solid test material is not used, direct correlation between thermodynamic aqueous solubility and kinetic solubility measured for a compound series is not often seen. However, it is considered that good solubility in a kinetic assay should facilitate the generation of reliable in vitro and in vivo data in drug discovery.

Shake Flask Method:

The equilibrium solubility of the un-ionized form of the samples was determined by the shake-flask method. It is based on simple procedures, but it is time consuming, and requires lots of manual work. But there is no accepted standard way to carry out this method ,because there are various parameters that effect the results but to attain equilibrium it is shaked for 24hrs-2 weeks.

Nogami Method:

Quite often a compound is rather unstable in aqueous solution .Hence long exposure to liquid required for traditional solubility measurement will cause decomposition and resulting solubility result will be unreliable .In such case Nogami method is used .In this method samples are taken at regular interval say at δ min ,from the shaking flask (supernatant is taken ).When amount dissolved at t+ δ min is plotted versus amount dissolved at t min ,a straight line is obtained .Such plot will give k as slope and putting those in Nogami equation will give the solubility .

C t+δ = S (1 – e -k δ) + e -k δ (Ct)

Where C is concentration ; S is solubility

δ is the time interval after which samples are taken

k is the constant .

PROCEDURE:

Preparation of Phosphate buffer-pH 6.8 (As per IP-2007):

50.0 ml of 0.2 M potassium dihydrogen phosphate was taken in a 200-ml volumetric flask. 22.4ml of 0.2 M sodium hydroxide was added and then water was added to make up the volume.

Calibration curve:

Solvent system: pH 6.8 Phosphate buffer solution

Stock solution: 10mg drug was dissolved in 100ml solvent system to get 100µg/ml stock solution.

Working standards: 2µg/ml, 4µg/ml, 6µg/ml, 8µg/ml, 10µg/ml, 12µg/ml solutions were prepared from stock solution

Calibration curve was prepared

Shake flask method:

To 5ml of pH 6.8 phosphate buffer solution, excess of drug was added.

The solution was kept for shaking in Orbital Shaker, the RPM was set to 300 for 24 hrsand is maintained at room temperature .

40µl of the supernatant was taken from the flask after 20, 40, 60, 80, 100, 120min & 24 hour And it was diluted further as follows to get the absorbance in the CC range.

Nogami method:

In this method the study was done only for 2hrs like shakeflask method , since it is used for those drugs which were unstable in the solvent system

OBSERVATIONS:

λmax was found to be 243nm from the spectra of working standard solution.

Calibration curve:

s.no Conc(µg/ml) absorbance1 2 0.17232 4 0.30843 6 0.41724 8 0.51425 10 0.6726 12 0.7838

0 2 4 6 8 10 12 140

0.10.20.30.40.50.60.70.80.9

f(x) = 0.0606471428571429 x + 0.0534533333333333R² = 0.99648550170723

calibration curve

Series2Linear (Series2)

conc(µg/ml)

Abso

banc

e

Shake flask method:

Dilutions:

40µl 10ml 300µl 10ml

Time (hr) absorbance

Conc(µg/ml)

Dilution factor

Microgram in 1 ml

Solubility mg/ml

0.33 0.1485 1.598333.3

13263.88 13.26388

0.66 0.1736 2.01 8333.3 16749.99 16.74999

1 0.1816 2.14 8333.3 17861.1 17.8611

1.33 0.21 2.61 8333.3 21805.55 21.80555

1.66 0.227 2.9 8333.3 24166.66 24.16666

2 0.2313 2.97 8333.3 24763.88 24.76388

240.2539 3.34 8333.3 27902.77 27.90277

0 5 10 15 20 25 300

5

10

15

20

25

30

SOLUBILITY VS TIME

TIME(hr)

SOL(

mg/

ml)

Here the study was done for 24 hrs and believed that it attains equilibrium during that time and the solubility was found to be 27.90mg/ml

Nogami method:

Dilutions:

40µl 10ml 300µl 10ml

Time in min absorbance c at t c at t+Δt y=x

00

013263.88

0

200.1485 13263.88 16749.99 13263.88

400.1736 16749.99 17861.1 16749.99

600.1816 17861.1 21805.55 17861.1

800.21 21805.55 24166.66 21805.55

1000.227 24166.66 24763.88 24166.66

1200.2313 24763.88

----24763.88

0 5000 10000150002000025000300000

5000

10000

15000

20000

25000

30000

f(x) = xR² = 1f(x) = 0.493063819207088 x + 12056.4018345642R² = 0.862317994060339

solubility by nogami method

Series2Linear (Series2)Series4Linear (Series4)

'C 'at t(µg/ml)

'C' a

t t+

tΔ(µ

g/m

l)

C(t+Δt)=S(1-e-kΔ)+e-kΔ(Ct) this is of form y=c+mx

e-kΔ(Ct)= mx = 0.493x k=0.0353

S(1-e-kΔ)= 12056

Solubility = S = 24807.9 µg/ml

Results:

Solubility of the given drug at room temperature in pH 6.8 Phosphate buffer was found to be:

By Nogami method: 24.807mg/ml

By Shake-flask method: 27.902mg/ml

References:

(1) Modern Pharmaceutics, Preformulation by Gilbert Banker and Chrishtopher Rhodes, 4th edition.

(2) “Study of equilibrium solubility measurement by saturation shake-flask method using hydrochlorothiazide as model compound” by Edit Baka et al , Journal of Pharmaceutical and Biomedical Analysis 46 (2008) 335–341.

(3) Study on standardization of shake-flask solubility determination method, by E.Baka, European journal of pharmaceutical sciences 325 (2007) 522–550