KINGDOM OF SAUDI ARABIA Laboratoual LABORATORY MANUAL WITH LOG BOOK BIOPHARMACEUTICS ... ·...

Laboratoual

L A B O R A T O R Y M A N U A L W I T H L O G B O O K

B I O P H A R M A C E U T I C S

P H T - 4 1 4

L E V E L I X

Course Cotor

Course Supervisor

Dr. Mohammad Javed Ansari

Email : [email protected]

A C A D E M I C Y E A R

1 4 3 8 - 1 4 3 9

PRINCE SATTAM BIN

ABDULAZIZ

UNIVERSITY

COLLEGE OF PHARMACY

DEPARTMENT OF PHARMACEUTICS

AL-KHARJ

KINGDOM OF SAUDI ARABIA

Name of the student:___________________________________

Student ID No:_______________________________________

mailto:[email protected]

DEPARTMENT OF PHARMACEUTICS LABORATORY MANUAL PHT 414

This laboratory manual will serve as

biopharmaceutical experiment protocol along

with logbook / homework / exercise.

You are supposed to complete the logbook /

homework / exercise on weekly basis.

There may be more than the prescribed

experiments to explore the understanding of

theory topics.

The list or content of experiment in the manual

may be substituted depending upon the

availability of chemical and instrument.

During experiment all the students need to

follow strict GMP/ GLP rules to avoid

occurrence of any untoward incidence.

PREFACE


Introduction & grading policy

Bio-pharmaceutics is the science that considers the interrelationship of the

physicochemical properties of the drug, the dosage form in which the drug is

given, and the route of administration on the rate and extent of drug

absorption. Thus it involves the study of the effects of dosage formulation on

drug absorption and distribution.

This course will provide the student with a basic understanding of bio-

pharmaceutics that can be applied to drug product development and drug

therapy.

Aims of the course are to familiarize students with the biological factors that

influence drug absorption; show how the physiochemical characteristics of the

drug influence absorption from the GIT; emphasize the importance of dosage

form selection and how it affects the clinical outcome; study the factors

affecting bioavailability of drugs including pharmacokinetics variability; study

the bio-pharmaceutics of sustained release and new drug delivery systems.

GRADING

Contents Grades Week

Experiment / QC & Lab discipline / indiscipline 5 Every week

Record completeness / homework 5 Every week

Lab Exam 10 13-14

Table Viva / Synopsis 5 13-14

Total 25

NECESSARY INSTRUCTIONS RELATED TO THIS COURSE

1. All students must complete all laboratory assignments. If a lab is missed,

the reason for the absence must be discussed with the instructor and a

makeup lab arranged attend the same in free time by discussion with

concerned supervisor.

2. Always bring with you: Scientific calculator with linear regression and

graph paper.

3. Submit the home work whenever instructed for the same.

4. The content of experiment or experiment partially or fully substituted

depending upon the availability of chemical and instrument.

5. There may be more than the prescribed experiments to explore the

understanding of theory topics.


Lab Indiscipline (-5)

EXP- DATE:

SECTION:

St Name Attendance Late entry

(-1)

No lab

coat

(-1)

Lab Indiscipline

(-1)

Dirty

Workplace (-1)

Dirty

Glass wares (-1)

Note: This form is to be kept with Lab instructor


Contents

Week Experiment, Exercise &

Preparation

QC/Lab

discipline /

Indiscipline

(5)

LAB

RECORD

(5)

Total

grades Sig.

1

Induction to Biopharma lab equipment

2 Aim: To determine of partition

coefficient of drugs

3

Aim: To determine the effect of

mixing time over partition coefficient

of drugs

4

Aim: To investigate the effect of pH

on the Partition Coefficient

(Absorption) of the drugs

5

Aim: To investigate the effect of pKa

on the Partition Coefficient

(Absorption) of the drugs

6

Aim: To investigate Absorption

behavior (Partition Coefficient) of

parent drugs and their salts.

7

Aim: To investigate effect of pH on

the absorption behavior (Partition

Coefficient) of neutral drug.

8 Aim: To investigate disintegration of

the dosage forms

9 Aim: To investigate effect of particle

size over dissolution of drugs

10 Aim: To investigate in vitro

transdermal diffusion of drugs.

11

Aim: To investigate effect of

formulation factor (unit strength) over

dissolution of drugs.


Experiment 1 Date: 26-09-2017

Student Name: Student ID:---------------------

Aim: To determine of partition coefficient of drugs

Principle and Theories

If a solute / drug is added to two immiscible liquids such as oil (organic phase) and

water (aqueous phase) in contact with each other, the solute / drug distributes itself

between the two liquids and an equilibrium is set up between the solute molecules in

oil and solute molecules in water (distributes or partitions between aqueous and oil

layers depending on solubility of drug in each layers). The ratio of the concentration

of the solute in the two liquids is known as distribution coefficient or partition

coefficient.

Partition Coefficient = [Concentration of drug in oil or organic phase] /

[Concentration of drug in water or aqueous phase]

Concentration of drugs in each layer can be evaluated by using several techniques

including High performance liquid chromatography (HPLC), UV spectrophotometry,

colorimetry, gravimetry, and titrimetric methods. Each method has its own merits and

demerits therefore these must be selected rationally.

Partition Coefficient of a drug is a measure of how well a substance distributes or

partitions between a lipid (oil) and water. High partition coefficient means more

tendency to distribute in lipids and less partition coefficient means less tendency to

distribute. Partition Coefficient in the range of 1 to 2 is supposed to predict passive

absorption of drug across lipidic membranes. High partition coefficient usually do not

result in more absorption as high lipid solubility and less water solubility may cause

precipitation of drug in the intestinal fluid. For optimum absorption, a drug should

have sufficient aqueous solubility to dissolve in the intestinal fluid at the absorption

site and lipid solubility high enough to facilitate partitioning of the drug in the lipoidal

membrane and into blood vessels.


Requirements

Solute or drug Citric Acid or ascorbic acid 0.2 M solution Water (aqueous phase)

Octanol or 2-Methylpropan-1-ol (isobutyl alcohol) (organic phase).

Sodium hydroxide (0.1 M NaOH), Phenolphthalein indicator

Separating funnel, titration apparatus, pipette etc.

Procedure

1. Add 25 ml of the 0.2 M Citric acid solution and 25 ml of 2-methylpropan-1-ol into a

100 ml separating funnel.

2. Stopper the funnel and shake vigorously for 6 minutes. (Release pressure in the

funnel

3. by occasionally opening the tap.)

4. Separate approximately 15 ml of each layer and collect them s e p a r a t e l y in

two clean beakers. (Discard the fraction near the junction of the two layers).

5. Pipette 5 ml of both liquids separately into titration flasks and titrate with 0.1 M

sodium hydroxide solution using 2 drops of phenolphthalein indicator.

6. Repeat the procedure three time and take average.

7. Calculate the ratio of the concentration of citric acid in the non-aqueous layer in

relation to the aqueous layer.

Observation and result

SN O = Volume of 0.1 M NaOH titre

for non-aqueous layer/ cm3

W= Volume of 0.1 M NaOH

titre for aqueous layer/ cm3

Partition coefficient (K)

O/W

1

2

3

Avg

Discussions:

1- Discuss the implication of different volume of NaOH consumed for aqueous

and non-aqueous layers.


2- Discuss the importance of partition coefficients of drugs in dosage form

design.




Aim: To determine the effect of mixing time over partition coefficient of drugs


If a solute / drug is added to two immiscible liquids such as oil (organic phase) and

water (aqueous phase) in contact with each other, the solute / drug distributes itself

between the two liquids and an equilibrium is set up between the solute molecules in

oil and solute molecules in water (distributes or partitions between aqueous and oil

layers depending on solubility of drug in each layers). The ratio of the concentration

of the solute in the two liquids is known as distribution coefficient or partition

coefficient.

Partition Coefficient = [Concentration of drug in oil or organic phase] /

[Concentration of drug in water or aqueous phase]

Concentration of drugs in each layer can be evaluated by using several techniques

including High performance liquid chromatography (HPLC), UV spectrophotometry,

colorimetry, gravimetry, and titrimetric methods. Each method has its own merits and

demerits therefore these must be selected rationally.

Partition Coefficient of a drug is a measure of how well a substance distributes or

partitions between a lipid (oil) and water. High partition coefficient means more

tendency to distribute in lipids and less partition coefficient means less tendency to

distribute. Partition Coefficient in the range of 1 to 2 is supposed to predict passive

absorption of drug across lipidic membranes. High partition coefficient usually do not

result in more absorption as high lipid solubility and less water solubility may cause

precipitation of drug in the intestinal fluid. For optimum absorption, a drug should

have sufficient aqueous solubility to dissolve in the intestinal fluid at the absorption

site and lipid solubility high enough to facilitate partitioning of the drug in the lipoidal

membrane and into blood vessels.


Requirements

Solute or drug Citric Acid or ascorbic acid 0.2 M solution Water (aqueous phase)




Procedure

Add 15 ml of the 0.2 M Citric acid solution and 15 ml of octanol into a 100 ml

separating funnel.

Stopper the funnel and shake vigorously for 5 minutes. Allow to stand for 5 minutes.

Collect 5 ml of top layer b y p i p e t t i n g a n d 5 m l o f b o t t o m l a y e r b y

o p e n i n g t h e k n o b o f f e n n e l a n d p o u r i n g i n m e a s u r i n g

c y l i n d e r .

T r a n s f e r b o t h s a m p l e s s e p a r a t e l y into two titration flasks, add 2 drops

of phenolphthalein indicator.and titrate with 0.2 M sodium hydroxide

solution and record the volume of NaOH consumed.

Add of 5 ml of drug aqueous solution and 5 ml of oil phase again in the same

separation funnel.

Repeat step 2, 3, 4 and record the reading

Calculate parti tion coefficient and compare the result .


Mixing

time

(min)

O = Volume of 0.1 M NaOH titre



titre for aqueous layer/ cm3


O/W

5

10

15

20

Discussions: Discuss some factors that can affect partition coefficients




Aim: To investigate the effect of pH on the Partition Coefficient (Absorption) of the

drugs


Effect of pKa on the partition of drugs can be explained by pH-partition theory in

conjunction with Henderson hasselbalch equation. Henderson hasselbalch equation

enables to find out extent of ionization at particular pH which in turn is dependent on

pKa of the drugs.

Acidic drugs having pKa equal to that of pH of medium (pKa - pH = 0) will be 50

% ionized i.e half ionized and half unionized.

Acidic drug having pKa slightly higher than that of pH of medium (pKa - pH =

0.5) will be 75 % ionized.

Acidic drug having pKa significantly higher than that of pH of medium (pKa – pH

> 1) will be 99-100 % ionized.

For Basic drugs: if pKa is by 2 units, 99% ionised

% ionization can be calculated using following formula.

Where X is 1 for acidic drugs while – 1 or basic drugs.

pH partition theory states that drugs are partitioned or distributed from the intestinal

fluid by passive diffusion depending on the fraction of neutral drug molecule /

unionized drug / undissociated drug at the pH of the intestine. Brodie et al. stated that

when a drug is ionized it will not be able to get through the lipid membrane, but only

when it is non-ionized and therefore has higher lipid solubility.

Thus, the process of portioning (absorption) will be affected by:

1. Partition Coefficient or the lipid solubility of the unionized drug.


2. Dissociation constant (pKa) of the drug.

3. pH of intestinal fluid (pH at absorption site).

Dissociation (Ionization) of drugs in aqueous solutions is a measure of the strength of

its acidity or a basicity and it is influenced by the pH of the medium.

For Weak Acids HA (aq) ↔ H+ + A

-

Dissociation constant of acid Ka = ( [H+][A-] / [HA] )

For many practical purposes it is more convenient to use or discuss the logarithmic

constant, pKa

where pKa = -log Ka,

or pKa = -log ( [H+][A

-] / [HA] )

or pKa = -log [H+] - log [A

-] / [HA] )

or pKa = pH - log [A-] / [HA] )

Lower the pKa of an acidic drug, stronger is the acid i.e. greater the proportion of

ionized form at a particular pH. Higher the pKa of a basic drug, the stronger is the

base. Thus, from the knowledge of pKa of drug and pH at the absorption site, the

relative amount of ionized and unionized drug in solution at a particular pH and the

percent of drug ionized at this pH can be determined by Henderson-Hasselbalch

equation:

or pH = pKa + log [A-] / [HA]

or pH = pKa + log [ionized drug] / [unionized drug]

pH – pKa = log [ionized drug] / [unionized drug]

log [ionized drug] / [unionized drug] = pH – pKa

[ionized drug] / [unionized drug] = antilog pH – pKa

[ionized drug] / [unionized drug] = 10 pH – pKa

Requirements

Solute or drug Citric Acid or ascorbic acid 0.1 M solution in different buffers (Simulated

gastric fluid, Simulated intestinal fluid, Acetate buffer)





Simulated gastric fluid: 2.0 g of sodium chloride and 3.2 g of purified pepsin (activity 800-

2500 units per mg of protein) in 7.0 mL of concentrated hydrochloric acid and water up to

1000 mL.

Simulated Intestinal Fluid: 6.8 g of monobasic potassium phosphate in 250 mL of water and

then adding 77 mL of 0.2 N sodium hydroxide and 500 mL of water. 10.0 g of pancreatin is

added and the resulting solution is adjusted with 0.2 N sodium hydroxide or 0.2 N

hydrochloric acid to a pH of 6.8 ± 0.1 and finally diluted to 1000 mL.

Saline pH 7.4, Phosphate-buffered Dissolve 2.38 g of disodium hydrogen orthophosphate ,

0.19 g of potassium dihydrogen orthophosphate and 8.0 g of sodium chloride in sufficient

water to produce 1000 ml and adjust the pH if necessary.

Acetate buffer: Acetate buffer is chosen to simulate the human skin pH condition of 5.5. To

prepare 1,000 ml of the acetate buffer solution, 150 g of sodium acetate was dissolved in ~250

ml of distilled water. Exactly 15 ml of glacial acetic acid was then added very slowly into the

sodium acetate aqueous solution. Finally, distilled water was added into the solution to fill the

volume.

Procedure

Prepare the buffer solutions to be studied and record their pH using pH meter.

Add desired amount of drug in the buffer solution to make 0.1 M

Transfer 10 ml of drug solution and add 10 ml of oil phase in 50 ml separating

funnel.

Stopper the funnel and shake vigorously for 5 minutes.

Allow to separate the layers.

Pipette 5 ml of both layers separately into titration flasks and titrate with 0.1 M


Calculate the ratio of the concentration of citric acid in the non-aqueous layer in

relation to the aqueous layer.

Repeat the procedure using different buffer solutions.

Compare partition coefficient of drug in aqueous solution with that of buffer

solution.


Observation and result for Ascorbic acid

Buffer

(pH)

O = Volume of 0.1 M NaOH titer



titer for aqueous layer/ cm3


O/W

Observation and result for Citric acid

Buffer

(pH)

O = Volume of 0.1 M NaOH titer





O/W

Discussions: Discuss the effect of pH on partition coefficients of drugs




Aim: To investigate the effect of pKa on the Partition Coefficient (Absorption) of the

drugs


Effect of pKa on the partition of drugs can be explained by pH-partition theory in

conjunction with Henderson HasselBach equation. Henderson Hasselbach equation

enables to find out extent of ionization at particular pH which in turn is dependent on

pKa of the drugs.

Thus, the process of portioning (absorption) will be affected by:

Partition Coefficient or the lipid solubility of the unionized drug.

Dissociation constant (pKa) of the drug.

pH of intestinal fluid (pH at absorption site).

Dissociation (Ionization) of drugs in aqueous solutions is a measure of the strength of

its acidity or a basicity and it is influenced by the pH of the medium.

For Weak Acids HA (aq) ↔ H+ + A

-

Dissociation constant of acid Ka = ( [H+][A-] / [HA] )

For many practical purposes it is more convenient to use or discuss the logarithmic

constant, pKa

where pKa = -log Ka,

or pKa = -log ( [H+][A

-] / [HA] )

or pKa = -log [H+] - log [A

-] / [HA] )

or pKa = pH - log [A-] / [HA] )

Lower the pKa of an acidic drug, stronger is the acid i.e. greater the proportion of

ionized form at a particular pH. Higher the pKa of a basic drug, the stronger is the

base. Thus, from the knowledge of pKa of drug and pH at the absorption site, the

relative amount of ionized and unionized drug in solution at a particular pH and the

percent of drug ionized at this pH can be determined by Henderson-Hasselbalch

equation:

pH = pKa + log [A-] / [HA]


or pH = pKa + log [ionized drug] / [unionized drug]

pH – pKa = log [ionized drug] / [unionized drug]

log [ionized drug] / [unionized drug] = pH – pKa

[ionized drug] / [unionized drug] = antilog pH – pKa

[ionized drug] / [unionized drug] = 10 pH – pKa

pH – pKa = log [unionized drug] / [ionized drug] (for basic drugs)

% ionization can be calculated using following formula.

Where X is 1 for acidic drugs while – 1 or basic drugs.






Requirements

Solute or drug Citric Acid or ascorbic acid 0.1 M solution in different buffers (Simulated

gastric fluid, Simulated intestinal fluid, Acetate buffer, phosphate buffer saline) (Aqueous

phase).






1000 mL.













volume.

Procedure


Add desired amount of drug in the buffer solution to make 0.1 M solutions


funnel.





Calculate the ratio of the concentration of citric acid or ascorbic acid in the non-

aqueous layer in relation to the aqueous layer.


Compare partition coefficient of drugs in aqueous solution with that of buffer

solution.

Observation and result for Ascorbic acid

Buffer (pH) O = Volume of 0.1 M NaOH

titer for non-aqueous layer/ cm3




O/W

SGF pH 1.2

SIF pH 6.8

PBS pH 7.4

ACETATE

BUFFER

pH 5.5


Observation and result for Citric acid

Buffer (pH) O = Volume of 0.1 M NaOH

titer for non-aqueous layer/ cm3




O/W SGF pH 1.2

SIF pH 6.8

PBS pH

7.4

ACETATE

BUFFER

pH 5.5

Discussions: Discuss the effect of pKa on partition coefficients of drugs

Discuss differences between Log P and Log D.




Aim: To investigate Absorption behavior (Partition Coefficient) of parent drugs and

their salts.


It has been evaluated that approximately 50% of all drug molecules marketed as

medicinal products are administered in a form of salts as they offer many benefits for

the pharmaceutical drugs. Salt formation is a relatively simple and powerful pre-

formation technique that can result in significant improvement of drug’s

physicochemical properties. Salts formation offer many advantages to the

pharmaceutical products as it can improve the solubility, dissolution rate,

permeability and efficacy of the drugs.

Salts are ionized in aqueous solutions giving cations and anions. Certain salts affect

the acidity or basicity of because some of the ions will undergo hydrolysis, The

general rule is that salts with ions that are part of strong acids or bases will not

hydrolyze, while salts with ions that are part of weak acids or bases will hydrolyze.

Consider NaCl. When it dissolves in an aqueous solution, it separates into Na+ ions

and Cl− ions:

NaCl → Na+(aq) + Cl−(aq)

Will the Na+(aq) ion hydrolyze? If it does, it will interact with the OH− ion to make

NaOH:

Na+(aq) + H2O → NaOH + H+(aq)

However, NaOH is a strong base, which means that it is 100% ionized in solution:

NaOH → Na+(aq) + OH−(aq)

The free OH−(aq) ion reacts with the H+(aq) ion to remake a water molecule:

H+(aq) + OH−(aq) → H2O

The net result? There is no change, so there is no effect on the acidity or basicity of

the solution from the Na+(aq) ion. What about the Cl− ion? Will it hydrolyze? If it

does, it will take an H+ ion from a water molecule:

Cl−(aq) + H2O → HCl + OH−


However, HCl is a strong acid, which means that it is 100% ionized in solution:

HCl → H+(aq) + Cl−(aq)

The free H+(aq) ion reacts with the OH−(aq) ion to remake a water molecule:

H+(aq) + OH−(aq) → H2O

The net result? There is no change, so there is no effect on the acidity or basicity of

the solution from the Cl−(aq) ion. Because neither ion in NaCl affects the acidity or

basicity of the solution, NaCl is an example of a neutral salt.

Things change, however, when we consider a salt like NaC2H3O2. We already know

that the Na+ ion won’t affect the acidity of the solution. What about the acetate ion?

If it hydrolyzes, it will take an H+ from a water molecule:

C2H3O2−(aq) + H2O → HC2H3O2 + OH−(aq)

Does this happen? Yes, it does. Why? Because HC2H3O2is a weak acid. Any chance

a weak acid has to form, it will (the same with a weak base). As some C2H3O2− ions

hydrolyze with H2O to make the molecular weak acid, OH− ions are produced. OH−

ions make solutions basic. Thus NaC2H3O2 solutions are slightly basic, so such a

salt is called a basic salt.






Requirements

Parent drug and its salt drug, 0.1 M solution in different buffers (Simulated gastric fluid,

Simulated intestinal fluid, Acetate buffer, phosphate buffer saline) (Aqueous phase).



Hydrochloric acid (0.1 M HCL), Phenolphthalein indicator




1000 mL.













volume.

Procedure


Add desired amount of parent drug or drug salt in the buffer solution to make 0.1 M

solutions


funnel.




sodium hydroxide solution (use 0.1 M HCL in case of drug salt) using 2 drops

of phenolphthalein indicator.

Calculate the ratio of the concentration of drug or salt in the non-aqueous

layer in relation to the aqueous layer.



solution.


Observation and result for Parent drug

Buffer (pH) O = Volume of 0.1 M NaOH titer for

non-aqueous layer/ cm3



Partition coefficient

(K)

O/W SGF pH 1.2

SIF pH 6.8

PBS pH 7.4

ACETATE pH 5.5

Observation and result for drug salt

Buffer (pH) O = Volume of 0.1 M HCL titer for

non-aqueous layer/ cm3

W= Volume of 0.1 M HCL


Partition coefficient

(K)

O/W SGF pH 1.2

SIF pH 6.8

PBS pH 7.4

ACETATE pH 5.5

Discussions: Discuss the effect of salt form of drug on absorption /partition

coefficients.




Aim: To investigate effect of pH on the absorption behavior (Partition Coefficient) of

neutral drug.


Neutral drugs are insensitive to litmus test as these do not ionize to give cations or

anions in aqueous solutions. Therefore, such drugs will remain mainly in molecular

form in the gastrointestinal tract. Which means that extent of absorption should be

similar in both acidic as well as basic pH of stomach and intestine respectively. Thus

prediction of absorption behavior of a neutral drug along gastro intestinal tract can be

envisaged by evaluating octanol-water partition coefficient of neutral drug using

different buffers in place of water.

Quantification of neutral molecules may be done by using analytical techniques such

as UV, HPLC, HPTLC. Which are examples of spectroscopic techniques that deals

with the study of interaction between Electromagnetic radiation and matter.

UV-Visible spectrophotometry is one of the most frequently employed technique in

pharmaceutical analysis. It involves measuring the amount of ultraviolet or visible

radiation absorbed by a substance in solution.

In qualitative analysis, organic compounds can be identified by use of

spectrophotometer, if any recorded data is available, and quantitative

spectrophotometric analysis is used to ascertain the quantity of molecular species

absorbing the radiation. Spectrophotometric technique is simple, rapid, moderately

specific and applicable to small quantities of compounds. The fundamental law that

governs the quantitative spectrophotometric analysis is the Beer -Lambert law.

Beer’s law: It states that the intensity of a beam of parallel monochromatic radiation

decreases exponentially with the number of absorbing molecules. In other words,

absorbance is proportional to the concentration.

Lambert’s law: It states that the intensity of a beam of parallel monochromatic

radiation decreases exponentially as it passes through a medium of homogeneous

thickness. A combination of these two laws yields the Beer-Lambert law.


Beer-Lambert law: When beam of light is passed through a transparent cell

containing a solution of anabsorbing substance, reduction of the intensity of light

may occur. Mathematically, Beer- Lambert law is expressed as

A=a b c

Where, A=absorbance or optical density

a=absorptivity or extinction coefficient

b=path length of radiation through sample (cm)

c=concentration of solute in solution.

The assay of single component sample, which contains other absorbing substances, is

then calculated from the measured absorbance by using one of three principal

procedures.

use of standard absorptivity value, calibration graph and single or double point

standardization. In standard absorptive value method, the use of standard A (1%, 1

cm) or E values are used in order to determine its absorptivity. It is advantageous in

situations where it is difficult or expensive to obtain a sample of the reference

substance. In calibration graph method, the absorbances of a number of standard

solutions of the reference substance at concentrations encompassing the sample

concentrations are measured and a calibration graph is constructed. The concentration

of the analyte in the sample solution is read from the graph as the concentration

corresponding to the absorbance of the solution. The single

point standardization procedure involves the measurement of the absorbance of a

sample solution and of a standard solution of the reference substance. The

concentration of the substances in the sample is calculated from the proportional

relationship that exists between absorbance and concentration.

Ctest= (Atest×Cstd)/Astd

Where Ctest and Cstd are the concentrations in the sample and standard solutions

respectively and Atest and Astd are the absorbances of the sample and standard

solutions respectively.



unionized drug / undissociated drug at the pH of the intestine.

https://www.omicsonline.org/open-access/standardization-of-clonal-macropropagation-protocol-of-dillenia-pentagyna-roxb-an-important-and-endangered-medicinal-tree-species-2155-952X-1000222.php?aid=74381


Requirements

Neutral drug. 0.1 % solution in different buffers (Simulated gastric fluid, Simulated intestinal

fluid, Acetate buffer, phosphate buffer saline) (Aqueous phase).





1000 mL.












volume.

Procedure


Add desired amount of neutral drug in water or in the buffer solution to make 0.1 M

solutions


funnel.



Pipette 1 ml of both layers separately and quantify the amount of drug present in

each sample by a suitable method. (dilute the samples if required).


Calculate the ratio of the concentration of neutral drug in the non-aqueous

layer in relation to the aqueous layer.



solution.

Observation and result for Parent drug

Buffer (pH) O = Absorbance of drug

in oil phase

O = Absorbance of drug

in water or buffer layers


O/W SGF pH 1.2

SIF pH 6.8

PBS pH 7.4

ACETATE pH 5.5

Discussions: Discuss the effect of pH on the on absorption /partition coefficients of

neutral drug




Aim: To investigate disintegration of the dosage forms

Theory and Principles

Disintegration is a process of disaggregation of dosage forms such as capsules or

compressed tablets into particles or granules. Disintegration does not imply complete

solution of the tablet or even its active constituent. It is performed to find out the time it

takes to disintegrate completely. Disintegration test is a measure of the quality of the oral

dosage form like tablets and capsules. Very high disintegration time means that the tablet

is too highly compressed or the capsule shell gelatin is not of pharmacopoeial quality.

Thus time of disintegration serves as a measure of the quality or performance of the

dosage forms. Moreover, it also serves as in process quality control test of the

formulation during formulation development and large scale production or

manufacturing. For instance, if the disintegration time is not uniform in a set of samples

being analysed, it indicates batch inconsistency and lack of batch uniformity. Thus

variables such as compression force, dwell time may be tuned to optimize the

formulations with the help of disintegration testing.

Pharmacopoeas have set standards for disintegration testing and the time required to

disintegrate completely. For instance, Uncoated and coated tablets should disintegrate in

15 and 30 minutes respectively. Complete disintegration is defined as the complete

disaggregation of dosage form identified as a state in which no residue remains on the

screen or if any residue of the tested dosage form remaining on the screen of the test

apparatus or adhering to the lower surface of the discs (if used) is a soft mass having no

palpably firm core (except fragments of insoluble coating or capsule shell).

Requirement

1- Marketed tablet or capsule formulation except states that are intended for use

as troches, or are to be chewed, or are designed as extended-release dosage

forms or delayed-release dosage forms.

2- Disintegration test apparatus: The apparatus consists of a basket-rack

assembly, a 1-litre beaker, a thermostatic arrangement for heating the fluid and


a mechanical device for raising and lowering the basket in the immersion fluid

at a constant frequency rate.

3- Disintegration media

4- Stop watch.

Procedure

1- Fill the basket with the disintegration media.

2- Switched on the apparatus after filling the water bath till mark.

3- Allow the thermostatic water bath to achieve a temperature of 37º ± 2 ºC.

4- Introduce one tablet or capsule into each tube and add a disc to each tube.

5- Operate the apparatus, using water maintained at 37º ± 2 ºC as the

immersion fluid unless another fluid is specified in the individual

monograph.

6- At the end of the time limit specified in the individual monograph, lift the

basket from the fluid, and observe the tablets for complete disintegration.

Note: If one or two tablets fail to disintegrate completely, repeat the test on 12

additional tablets: not less than 16 of the total of 18 tablets tested disintegrate

completely.

7- If the tablets or capsules adhere to the disc and the preparation under

examination fails to comply, repeat the test omitting the disc. The preparation

complies with the test if all the tablets or capsules in the repeat test

disintegrate.

Observation and Results

Trial Formulation Specified Time Result


Discussions: Discuss the importance of disintegration test.




Aim: To investigate effect of particle size over dissolution of drugs


Physicochemical property of drug candidate plays very important role in the

performance of final finished product which is usually measured in terms of

bioavailability. Bioavailability of orally administered product is end result of dosage

forms after successful administration-dissolution-absorption of drugs form the dosage

forms. Thus performance or bioavailability of oral dosage forms depends on how well

drug is administered, dissolved and absorbed. Solutions/ syrups are rapidly absorbed

as drug is already dissolved, however, rate of absorption of drugs from tablets, capsule

and suspension is dependent of the rate of dissolution of drugs from these dosage

forms. Thus rate of dissolution often becomes rate limiting step in the

absorption/bioavailability or performance of such dosage forms. Dissolution tests are

used in the pharmaceutical industry for several purposes.

1- Dissolution test is helpful in drug development process.

2- Dissolution test can be used as a prognostic tool of oral drug absorption or

bioequivalence.

3- Dissolution test can be used as a quality control test for final formulations.

4- Dissolution test can be a tool for in vitro in vivo correlation of drug.

Dissolution is transfer of mass from a solid to a liquid phase that involve liberation of

drug molecules from the solid phase at solid liquid interface (boundary), then

transport of solute molecules to the bulk liquid due to diffusion. Noyes and Whitney

described the dissolution process quantitatively. Mathematical expression of Noyes

and Whitney law-

dc/dt= K (Cs- C)

Where, dc/dt= rate of dissolution,

Cs= saturation solubility of the substance,

C= concentration at the expiration of the time t, and

K=dissolution constant.


Mayer and Whitney (modified Noyes Whitney equation) states that rate of dissolution

depends upon the surface area of the solid, thus finely divided drug will dissolve faster

due to higher surface area.

dc/dt= KS (Cs- C)

Dissolution constant (K), is equal to (D/h), the ratio of diffusion coefficient and

thickness of diffusion layer (a stationary film of saturated drug solution adjacent to the

surface of solid). In dissolution rate limited absorption, C is negligible therefore

equation may be written as follow

dc/dt= DSCs / h

Thus dissolution (rate of mass transfer) of drug will depend on-

1- Diffusion coefficient of drug

2- Surface area of the formulation

3- Concentration of drug in the formulation

4- Thickness of diffusion layer

Effect of surface area of the formulation can be evaluated by comparing the

dissolution profiles of equal amount of two drug samples but with different particle

sizes.

Requirements Coarse drug powder

Micronized drug powder

Dissolution media

Dissolution apparatus

UV spectrophotometer

Glasswares and pipettes

Procedure

Place 900 ml of dissolution medium in the dissolution vessel then equilibrate the

dissolution medium to 37 + 0.5 o C.

Prepare the drug particles to be studied by micronizing it for 5 minutes.

Place equal amount of coarse powder and micronized powder in suitable size

capsule cell.

Run the apparatus and withdraw the samples at specified times with replacing with

fresh medium.

Dilute samples appropriately then perform the analysis by UV samples.


Plot the concentration versus time graphs and compare.


Time

(min)

Absorbance of micronized powder

Absorbance of coarse powder

0

5

10

15

20

25

30

Discuss the effect of particle size over dissolution of the drug




Aim: To investigate in vitro transdermal diffusion of drugs.


In vitro transdermal diffusion studies are undertaken to study the amount of drugs

absorbed / permeated from a topically applied transdermal drug delivery system such

as ointment, cream, gels or transdermal patches through human / animal skin or

artificial membranes.

The most common technique employs an open chamber design like the Franz

diffusion cell consisting of a donor and receiver compartments. The skin or synthetic

membrane separates the donor compartment containing the test product from the

receptor compartment that is filled with diffusion medium such as phosphate buffered

saline (PBS), phosphate buffers for water soluble drugs however, hydrophobic drugs

should be studied in hydro alcoholic solutions like isopropanol/PBS-50/50 (v/v).

Diffusion medium capable of provide sink conditions i.e. high capacity to dissolve or

carry away the drug along with frequent sampling and fresh media replacement are

very important considerations in the diffusion test. Concentration of drug in the

diffusion media /receptor media should not exceed 10% of Cs (drug solubility in the

releasing matrix) at the end of the test. Usually five-six time points over an

appropriate time period like at 30 min, 1,2,4 and 6 hr are suggested to get an adequate

release profile or diffusion profile. However it can be extend upto 24 hr depending on

the clinical use and desired experimental information. Samples are analyzed by

appropriate analytical methods and plotted against time or square root of time to

construct release rates graphs (μg/hour) or permeation graphs (μg/cm2 ∙ √hr). In

addition to reporting release rates, other values, such as the API’s flux (μg/cm2 hr),

24-hr accumulation (μg/cm2) and permeability (cm/hr) can also be reported so that a

more complete picture of API release and accumulation can be generated. The slope

of the linear portion of early time release, i.e., the steady state presents the flux of the

API, and the Y-axis labels the accumulation of API at certain time. Permeability (P),

defined as the rate of flow of drug through a porous material such as membrane, is

calculated by the following equation:


Requirements

Formulation

Diffusion media (Phosphate buffer saline)

Diffusion membranes

Diffusion apparatus (Franz diffusion cell)



Procedure

The apparatus consists of an automatic Franz diffusion cell or alternatively a

cylindrical open ends glass or plastic tube.

Artificial membrane (previously soaked in receptor medium for overnight – 24

hours) is fixed to the donor compartment of diffusion cell or alternatively one end

of tube.

One gram of transdermal formulation is uniformly spread on the surface of

membrane such that the preparation occupies inner circumference of the

membrane.

Diffusion medium is filled in receiver compartment of Franz cell or alternatively

approximately 40 ml is taken in a 50 ml beaker kept on thermostatic hot plate

maintained at 37±2 0C with magnetic stirrer stirring at 50 rpm.

Donor compartment is mounted over receiver compartment or alternatively tube is

mounted in the diffusion medium such that the lower end of tube containing

formulation is touching the surface of diffusion medium.

Samples are withdrawn at definite interval and the same amount is replaced with

fresh medium each time.

Samples are diluted appropriately then analyzed by UV method.


Time

(min)

Absorbance Concentration Cumulative

amount

Permeability of

drug

10

20

30

60

120


Discussions: Discuss the factors affecting transdermal diffusion of drugs




Aim: To investigate effect of formulation factor (unit strength) over dissolution of drugs.


Formulation factors such as types of formulation, unit size of formulation plays very

important role in the performance of the product which is usually measured in terms

of bioavailability. Bioavailability of orally administered product is end result of

dosage forms after successful administration-dissolution-absorption of drugs form the

dosage forms. Thus performance or bioavailability of oral dosage forms depends on

how well drug is administered, dissolved and absorbed. Solutions/ syrups are rapidly

absorbed as drug is already dissolved, however, rate of absorption of drugs from

tablets, capsule and suspension is dependent of the rate of dissolution of drugs from

these dosage forms. Thus rate of dissolution often becomes rate limiting step in the

absorption/bioavailability or performance of such dosage forms. Dissolution tests are

used in the pharmaceutical industry for several purposes.

1- Dissolution test is helpful in drug development process.

2- Dissolution test can be used as a prognostic tool of oral drug absorption or

bioequivalence.

3- Dissolution test can be used as a quality control test for final formulations.

4- Dissolution test can be a tool for in vitro in vivo correlation of drug.

Dissolution is transfer of mass from a solid to a liquid phase that involve liberation of

drug molecules from the solid phase at solid liquid interface (boundary), then

transport of solute molecules to the bulk liquid due to diffusion. Noyes and Whitney

described the dissolution process quantitatively. Mathematical expression of Noyes

and Whitney law-

dc/dt= K (Cs- C)

Where, dc/dt= rate of dissolution,

Cs= saturation solubility of drug (concentration of diffusion layer),

C= concentration of drug in the dissolution medium at time t.

K=dissolution constant.


Mayer and Whitney (modified Noyes Whitney equation) states that rate of dissolution

depends upon the surface area of the solid, thus finely divided drug will dissolve faster

due to higher surface area.

dc/dt= KS (Cs- C)

Dissolution constant (K), is equal to (D/h), the ratio of diffusion coefficient and

thickness of diffusion layer (a stationary film of saturated drug solution adjacent to the

surface of solid). In dissolution rate limited absorption, C is negligible therefore

equation may be written as follow

dc/dt= DSCs / h

Thus dissolution (rate of mass transfer) of drug will depend on-

1- Diffusion coefficient of drug

2- Surface area of the formulation

3- Concentration of drug in the formulation

4- Thickness of diffusion layer

Effect of unit strength of formulation over its performance can be investigated by

comparing dissolution profiles of equal amount of two drug samples with different

unit sizes.

Requirements

Tablets with different unit strengths

Dissolution media

Dissolution apparatus




Procedure

Place 900 ml of dissolution medium in the dissolution vessel then equilibrate the

dissolution medium to 37 + 0.5 o C.

Place equal dosage of tablets with different unit sizes in separate dissolution flask

Run the apparatus and withdraw the samples at specified times with replacing with

fresh medium.

Dilute samples appropriately then perform the analysis by UV samples.

Plot the concentration versus time graphs and compare.


Time

(min)

Absorbance of 100 mg tablets

Absorbance of 50 mg tablet + 50 mg tablets

0

5

10

15

20

25

30

Discussions: Discuss the effect of unit size over dissolution of the drug

KINGDOM OF SAUDI ARABIA Laboratoual LABORATORY MANUAL WITH LOG BOOK BIOPHARMACEUTICS ... ·...

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