Biopharmaceutics and Pharmacokientics A Pracitcal Manual Compiled by Deepa MK

Biopharmaceutics
& Pharmacokinetics
A Practical Manual
Compiled By: Deepa MK,M.Pharm.,

Assistant Professor,
Department of Phrmaceutics,
Nehru college of Pharmacy,
Thiruvilwamala, Thrissur
INDEX
Sl . Date Experiments Page No. Marks Sig.

No.
Data Analysis
1 Data Analysis I
2 Data Analysis II
3 Data Analysis III
4 Data Analysis IV
5 Data Analysis V
6 Data Analysis VI
Experiments
7 Determination of Partition Coefficient And Effect If
pH on Partition Coefficient of Salicylic Acid
8 Demonstration Of In Vitro One Compartment

Model After Administration Of Single IV Bolus
Dose Simulating Plasma Elimination And Urine
Excretion
9 Studies On Protein Binding Of Salicylic Acid
10 Diffusion Study Of Salicylic Acid By Using Egg

Membrane
11 In Situ Drug Absorption Study Of aspirin tablet By

Using Everted Chicken Ileum
12 Formulation And Evaluation Of Transdermal Patch

Of Paracetamol Using HPMC
13 Dissolution Study of Uncoated Marked Tablets

CALCULATIONS
Time Plasma Plasma

Concentration Concentration
( hrs) (µg/ml ) X Time
0 0
0.5 5.4
1 10
2 17.2
4 25.8
8 29.6
12 26.6
18 19.4
24 13.3
36 5.9
48 2.6
72 0.5
Laboratory Manual of Biopharmaceutics and Pharmacokinetics 1

Exp.No:1 Date:
DATA ANALYSIS-I
AIM
To determine the pharmacokinetic parameters such as Cmax, tmax, KE, , t ½, ,

.
, , ,MRT , from the given data
DATA
Drug concentration in plasma, following oral administration of a fully absorbed 500mg dose
of a drug is given below.
Concentration
Time
(µg/ml )
( hrs)
0 0
0.5 5.4
1 10
2 17.2
4 25.8
8 29.6
12 26.6
18 19.4
24 13.3
36 5.9
48 2.6
72 0.5

1. Cmax : Peak plasma level computed directly from the plasma level profile.
2. t max: Time to achieve peak plasma level computed directly from the plasma level
profile.
3. Elimination rate ( KE):Elimination rate constant is calculated from the terminal

elimination phase of log plasma concentration Vs time by least square regression analysis. K E
is calculated as KE = 2.303 × Slope
4. Biological Half-life ( t 1/2)
t½
5.
The extent of absorption is calculated from area under the plasma concentration is calculated
from area under the plasma concentration- time profile from zero to t* by trapezoidal rule.
6.
The estimation of area under the blood level time curve from zero time to infinity must be
carried out in 2 steps.
1. The area under the curve from zero to time t* is calculated by means of trapezoidal
rule.
2. The area under curve from t* to infinity by using formulae
where = 2.303 times the slope of terminal exponential phase of a plot of log
plasma concentration Vs time
t* = time at which blood sampling is stop
C* = concentration of drug at t*

7.
Plot of the product of concentration and time Vs time from zero time to t* is often
referred to as the area under the first moment curve.
8.
The estimation of area under first moment curve from zero time to infinity must be carried
out in 2 steps.
1. The area under first moment curve from zero to time t* is calculated by means of
trapezoidal rule.
2. The area under first moment curve from t* to infinity by using formulae
= 2.303 times the slope of terminal exponential phase of a plot of log plasma
concentration Vs time
t* = time at which blood sampling is stop
C* = concentration of drug at t*
9. Mean Residence Time ( MRT ):it is defined as the average amount of time spent by the
drug in the body before being eliminated.
MRT =

REPORT
1. Cmax
2. t max
3. KE
4. t 1/2
5.
6.
7.
8.
9. MRT
REFERENCES
1.P L Madan. Biopharmaceutics and pharmacokinetics.1st edition; p.196-197
2. C.Vijayaraghavan ,Experimental biopharmaceutics and pharmacokinetics ,1st edition. P.61-

64

Expt.No:2 Date:
DATA ANALYSIS-II
AIM
To determine the pharmacokinetics parameters like KE , Vd,and C0 following 250mg

IV bolus administration of one compartment kinetics.
DATA
Plasma
Time (hr) Concentration (µ/ml)
1 8
2 6.3
3 4.9
4 3.9
5 3.1
6 2.5
7 1.9
THEORY
In one compartment model, the body is considered as single, kinetically homogenous

unit that has no barriers to movement of drugs and final distribution equilibrium between the
drug in plasma and other body fluid is attained instantaneously and maintained at all times.
This method thus applied only to those drugs that distribute throughout the body.

One compartment model-Intravenous Bolus Administration
When a drug that distributes rapidly in the body is given in the form of a rapid
intravenous injection (i.e. i.v. bolus or slug), it takes about one to three minutes for complete
circulation and therefore the rate of absorption is neglected in calculations. The model can be
depicted as follows:
Blood and other

Body tissue
The general expression for rate of drug presentation to the body is
= Rate in (availability) – Rate out (elimination)------------------------ (1)
Since rate in or absorption is absent, the equation becomes;
= -Rate out ------------------------------------------------------------------(2)
If the rate out or elimination follows first-order kinetics, then:
= -KE X -----------------------------------------------------------------------(3)
Where KE = first-order elimination rate constant, X = amount of drug in the body at any time
t remaining to be eliminated and negative sign indicates that the drug is being lost from the
body. The various related pharmacokinetic parameters can now be estimated.
Elimination rate constant (KE)
For a drug that follows one compartment and administered as IV bolus injection, the decline
in plasma drug concentration is only due to elimination of drug from body ,the phase being
called as elimination phase.
Elimination half-life(t ½)
It is the time taken for the amount of drug in body as well as plasma concentration to decline
by one half or 50%of its initial value .in case of elimination process, biological half-life ie

Apparent volume of distribution (Vd)
It is the measure of extent of distribution of drug and is expressed in litre.
Vd = X0/C0
Where X 0 is dose administered, C0 is initial plasma concentration.
REPORT
Elimination rate constant (KE) =
Volume of distribution (Vd) =
Elimination half-life (t1/2) =
Initial concentration (C0) =
REFERENCES
1.D.M Brahmankar, Sunil B. Jaiswal, Biopharmaceutics and Pharmacokinetics A Treatise,

First edition , Vallabha Prakashan, Delhi, 2006, P.290 - 292

Expt:3 Date:
DATA ANALYSIS-III
AIM
To determine absorption rate constant from given oral absorption data given by using
method of residuals.
DATA
Time Plasma
Concentration
( hrs) (µg/ml )
1 0.38
2 0.73
3 0.91
4 0.97
5 0.97
6 0.92
8 0.71
10 0.53
12 0.40
14 0.30
THEORY
The absorption rate constant can be calculated by methods of residuals and the technique is
also known as feathering,peeling,,stripping or curve fitting.it is commonly used in
pharmacokinetics to resolve a multi exponential curve in to its individual components. For a
drug that follows one compartment kinetics and administrated orally, the concentration of
drug in plasma is expressed by,

CALCULATIONS:
Extrapolated Residual
Time Concentration Concentration Value
( hrs) (µg/ml )
(µg/ml )
1 0.38
2 0.73
3 0.91
4 0.97
5 0.97
6 0.92
8 0.71
10 0.53
12 0.40
14 0.30

- ----------------------------------------------(1)
, a hybride constant ,then
C = - ----------------------------(2)
During the elimination phase, when absorption is almost over Ka>>KE and the value of 2nd
exponential approaches zero whereas, first exponential retains some finite

value.
At this time equation (2 )reduces to,
-------------------------------(3)
In log form above equation is
-----------------------------(4)
Where C Represents the back extrapolated plasma concentration values.a plot of log c
versus t yields a biexponential curve with a linear phase having slope –KE/2.303,back
extrapolation of this straight line to time zero yields y-intercept equal to log A.
Subtraction of true plasma concentration value i.e. equation.(3) from equation (4) yields a
series of residual concentration values Cr.
-----------------------(5)
In log form, the equation is:
----------------------(6)

A plot of log Cr Versus t yields a straight line with slope -Ka/2.303 and y-intercept logA.
Absorption half-life can then be computed from Ka using the relation 0.693/Ka. Thus,
the method of residuals enables resolution of biexponential plasma level-time curve into
its two exponential components.
Solution:
1.plot plasma drug concentration on Y-axis time and on x-axis in a semilogarithmic graph
paper.
2.back extrapolate the log linear of residual portion of the decline phase. Let ‘C’ denotes the
plasma concentration alone this extrapolated line.
3.sustract the absorbed plasma concentration (C) from corresponding extrapolated value at
each time point.
4.plote the residual (C- C ) against time on the same semilogarithemic graph paper.
If the residual plot is an straight line ,then the absorption is 1st order process.the slope is equal
to-Ka/2.303,
Ka=slope ×2.303.
REPORT
The absorption rate constant from the given oral absorption data was found to be
REFERENCES
1. C.Vijayaraghavan,Experimental biopharmaceutics and pharmacokinetics 1st edition ,P.113-115

Expt No: 4 Date:
DATA ANALYSIS-IV
AIM
To determine absorption rate constant, elimination rate constant, Tmax, Cmax and
volume of distribution from given data.
DATA
Drug concentration in plasma at different time interval following oral admiration of
100mg of drug is given below by assuming that administrated dose is fully absorbed.
Time Plasma
Concentration
( hrs) (µg/ml )
0.25 1.6
0.5 2.7
1 3.7
2 3.5
3 2.7
4 2
6 1.02
8 0.49
10 0.26
12 0.12

CALCULATIONS:
Plasma Extrapolated Residual

Time
Concentration Concentration Value
( hrs) (µg/ml ) (µg/ml )
0.25 1.6
0.5 2.7
1 3.7
2 3.5
3 2.7
4 2
6 1.02
8 0.49
10 0.26
12 0.12

REPORT
Absorption rate constant :
Elimination rate constant:
Tmax :
Cmax :
Volume of distribution :
REFERENCES
1.S.B.Bhise, R.J.Dias et.al, Laboratory manual of biopharmaceutics and

pharmacokinetics, first edition, Trinity publishing house, Satara, p.132

Expt .No: 5 Date:
DATA ANALYSIS-V
AIM
To determine elimination rate constant and excretion rate constant from given urinary
excretion data by excretion rate method.
DATA
Time of urine Volume of Concentration

collection(hr) urine of unchanged
Sample collected(ml) drug in
urine(mcg/ml)
1 0-2 140 250
2 2-4 150 100
3 4-6 90 80
4 6-8 20 20
5 8-12 310 10
6 12-24 600 4

CALCULATIONS:
Time of Volume Concentrati Urine midpoint amount excretio

urine of urine on of Collectio of urine excreted n rate
collection collected unchanged n Interval collection in tine
Sample drug in (ðd) interval (mg/hr)
(hr) (ml) urine(mcg/ (t*)
ml) (mg)
1 0-2 140 250
2 2-4 150 100
3 4-6 90 80
4 6-8 20 20
5 8-12 310 10
6 12-24 600 4

REPORT
Elimination rate constant :
Excretion rate:
REFERENCES
1. D.M Brahmankar, Sunil B. Jaiswal, Biopharmaceutics and Pharmacokinetics A Treatise,

First edition , Vallabha Prakashan, Delhi, 2006, P.

Expt No:6 Date:
DATA ANALYSIS-VI
AIM
To determine elimination rate constant from given urinary excretion data by Sigma
Minus method.
DATA
Time of urine Volume of Concentration

collection(hr) urine of unchanged
Sample collected(ml) drug in
urine(mcg/ml)
1 0-2 140 250
2 2-4 150 100
3 4-6 90 80
4 6-8 20 20
5 8-12 310 10
6 12-24 600 4

CALCULATIONS
Time Volu Concen Urine midpoint amou excretio Cumula Amou

of me of tration Collec of urine nt n rate tive nt
urine urine of tion collectio excre amount reami
Samp collect collect unchan Interv n ted in (mg/Hr) excrete ng to
le ion ed ged al tine d be
drug in (ðd) (t*) inter excret
(hr) (ml) urine( val (mg) e
mcg/ml
) (mg)
(mg)
1 0-2 140 250
2 2-4 150 100
3 4-6 90 80
4 6-8 20 20
5 8-12 310 10
6 12-24 600 4

REPORT
The elimination rate constant was found to be _________________from the given

urinary excretion data by Sigma Minus method.
REFERENCES
1.D.M Brahmankar, Sunil B. Jaiswal, Biopharmaceutics and Pharmacokinetics A Treatise,

First edition , Vallabha Prakashan, Delhi, 2006, P.

Expt No:7 Date:
DETERMINATION OF PARTITION COEFFICIENT AND EFFECT OF pH ON THE

PARTITION COEFFICIENT OF SALICYLIC ACID
AIM
To determine the partition coefficient of salicylic acid and effect of PH on partition

coefficient of salicylic acid.
MATERIAL REQUIRED
Salicylic acid, n-octanol, potassium dihydrogen phosphate, FeNO3 solution, colorimeter

separating funnels.
PRINCIPLE
According to pH partition hypothesis the gastrointestinal epithelial act as a lipid

barrier towards drug which are absorbed by passive diffusion and those that are lipid soluble
will pass across the barrier as much drugs are weakly acidic or basic drug will pass across the
gastro intestinal epithelia is impermeable to the ionized form of some drug. The extent to
which a weakly acidic drugs or basic drugs ionized in solution in the gastro intestinal fluid
may be calculated by using HINDERSON HESSELBATCH equation. Drugs having a single
ionisable group [Aspirin/ salicylic acid]. The equation takes the focus on
Log [A]/[HA] = pH = PKa
Where
 PKa = negative logarithm of acid dissolution constant

 [HA] = concentration of unionized form
 [A] = concentration of ionized form
 PH =PH of GIT tract
For week basic drug log[BH]/[B]=PKa-pH
Where
 [BH]=concentration of un ionized form

 [B] =concentration of ionized form
According to the equation of weakly acidic drug PKa-3.6 will be

predominantly consider at GI fluid at PH 1.2(98.4%) and almost totally considered in
intestinal fluid at pH 6.8(98.8%) where a weakly basic drug PKa 5 will almost entirely
consider (99.8%) at gastric pH or 6.8(98.4%)

PROCEDURE
Preparation Of Standard Graph Of Salicylic Acid
1. 100 mg of salicylic acid is weighed and transfered into 100ml standard flask and make up
the volume upto 100mi using distilled water(stock solutionA)
2. From the stock solution a various strength of concentration like 20,40,60,80&100
microgram per ml are prepared by taking 2,4,6,8&10ml from the stock solution A and
diluting upto 100 ml by using distilled water
3. From corresponding concentrations pipette out 5 ml and add 2.5mi of 4 % FeNO3
solution
4. Measure the absorbance at 547nm by means of colorimeter. Standard graph is plotted by
taking concentration of salicylic acid on X axis and the absorbance at 547nm on Y axis
Determination Of Partition Coefficient
1) Four separating funnels are taken clean thoroughly and dry

2) The separating funnels are named as F1,F2,F3&F4
3) 25ml o0f 2.2 pH buffer is added and 25 ml of n-octanol is added to separating funnel F1
100mg of salicylic acid is added to the separating funnel and allow to separate & 1ml of
aqueous layer is removed and transferred into standard flask and make upto 100ml From
this solution 5ml is taken and add 2.5 ml of 4% FeNO3 solution is added. The absorbance
is measured at 547 nm by taking 2.5ml of FeNO3 solution and 5ml water as blank
4) Take 25ml of n-octanol and 25ml of pH4 buffer solution and procedure is repeated
at pH 7.2& 8 in separating funnels F3 & F4
5) The absorbance at different pH and concentration of salicylic acid are taken and noted
down from the standard graph and the value is multiplied by multiplying factor
REPORT Partition coefficient of given drug of salicylic acid and effect of pH on

partition coefficient was noted
 Partition coefficient at pH 2.2 =

 Partition coefficient at pH 4 =
 Partition coefficient at pH 7.2 =
REFERENCES
. C.Vijayaraghavan and Judith Justin. Experimental biopharmaceutics and pharmacokinetics 1 st edition ,P.4-9

Expt No:8 Date:
DEMONSTRATION IN VITRO ONE COMPARTMENT MODEL AFTER

ADMINISTRATION OF SINGLE IV BOLUS DOSE SIMULATING PLASMA
ELIMINATION AND URINE EXCRETION
AIM
To study in vitro compartment modeling after administration of single IV dose

simulating plasma elimination and urinary excretion.
MATERIALS REQUIRED
Glass beakers, pipettes, test tubes, connecting tubes, UV -Vis Spectrophotometer etc.
PRINCIPLE
By an arrangement of beakers and a constant head water reservoir, it is possible to

simulate plasma concentrations and drug amounts in urine after IV bolus administration. The
constant flow of water through the system causes a first order kinetics. In this experiment
Salicylic acid is used as a model compound and its various pharmacokinetic parameters are
obtained by simulating the plasma elimination and urinary excretion after giving a single IV
bolus dose. The drug concentration can be determined spectrophotometrically at 547 nm, by
sampling the water from plasma compartment (P) and urine compartment (U) periodically.
The pharmacokinetic parameters of the system can be determined by analyzing the

obtained data.The plasma concentration versus time is plotted on semilog paper and the
parameters; elimination rate constant, elimination half life, plasma concentration at zero time
(C0 ), volume of distribution, clearance and area under curve can be determined. The rate of
excretion versus time is plotted on semilog paper and parameters elimination rate constant,
excretion rate constant and elimination half life can be determined.
Models are used to describe and interpret a set of data obtained by experimentation.
Pharmacokinetic model provide concise means of expressing mathematically or
quantitatively, the time course of drug throughout the body and compute meaningful
pharmacokinetic parameters. Three different approaches to pharmacokinetic analysis for
experimental data are compartment modeling, non-compartmental analysis, physiologic
modeling. A compartment is an entity, which can be described by definite volume and
concentration.
In the case of IV bolus administration the drug distributes rapidly in the body. The
disposition of a drug follows one compartment kinetic model and it is monoexponential and
the equation is
Dв= Dв0. E‫־‬kEt (or) log Dв = -kEt/2.303 + log Dв0
Dв = Amount of Drug in the body.
Dв0= Amount of drug at zero time.
KE = Elimination rate constant.
It is a pharmacokinetic parameter that permits the use of plasma drug concentration (Cp) in
the place of Dв. In terms of volume of distribution.
Cp0= Dвº/Vd (or) Vd= Dвº/Cp0
DIAGRAM
Design of apparatus to simulate one compartment open model IV bolus dose
EXPERIMENTAL PROCEDURE
 Set the plasma (beaker) and urine (measuring cylinder) components to the
apparatus.
 Arranged such that the buffer is continuously pumping to the central compartment
( beaker) through peristaltic pump.

 Set the beaker on the magnetic stirrer and adjusted for gentle mixing.
 Established a flow rate of 8.4 ml/ minute.
 Injected the plasma (beaker with the dose of drug salicylic acid (100mg).
 Placed an empty measuring cylinder in position to collect the urine sample used
buffer (pH 7.4 phosphate buffer) as the blank.
 Plasma sample: Collect (1ml) plasma sample at 0,5, 10, 15, 30,45, and 60 min
after the dose. The test tubes were arranged and labeled to accommodate the
samples. Diluted 1ml sample to 10ml with buffer.
 Urine sample:Urine samples (1ml) were collected at 0,5, 10, 30, 45, and 60
minutes. At each time point for unit collection replace the beaker with empty
beaker. Measured the volume collected, mixed the contents of beaker to obtain a
uniform solution and diluted 1ml sample to obtain a uniform solution and diluted
1ml sample to 10ml for analysis. Each sample was analyzed using colorimeter at
547 nm.
 Data Analysis:. Calculated Ke from urinary excretion data. Plot concentration vs
time on semi log graph paper and calculate KE from the slope, half life, Volume of
distribution, initial concentration and clearance.
REPORT
Elimination rate constant, (KE) =
Excretion rate constant, (Ke) =
Volume of distribution, (Vd) =
Initial plasma drug concentration, (C0) =
REFERENCES
1.Leon Shargel et.al, Applied Biopharmaceutics and Pharmacokinetics. 5th edition; Mc Graw
Hill Companies, Singapore, 2005, P. 51 - 70

Exp.No:9 Date :
STUDIES ON PROTEIN BINDING OF SALICYLIC ACID
AIM
To determine the percentage of salicylic acid that undergoes protein binding across a
semipermable membrane.
REQUIREMENTS
Salicylic acid, egg albumin, ferric nitrate, hydrochloric acid, egg membrane, and distilled
water. standard flask, beaker, open end tube UVspectrophotometer, magnetic stirrer,
PRINCIPLE
A drug in the body can interact with several tissue components of which two major
categories are blood and extra-vascular tissues. The phenomenon of complex formation of
drugs with protein is called protein binding of drugs. Binding to Plasma Proteins like
Albumin affects drug distribution and elimination as well as the pharmacological effect of the
drug. Only that fraction of the drug concentration that is freely circulating or unbound can
penetrate cell membrane and is subjected to glomerular filtration. The most important
contribution to drug binding in the plasma is made by albumin, which comprises. About half
of total the plasma proteins, albumin binds a wide variety of drug molecules but plays an
important role binding of weak acids and neutral drugs.
In this experiment, Tube A contains only Salicylic acid with no protein in it. The
equilibrium of Salicylic acid in tubeA and beaker is achieved through semipermeable
membrane and the concentration of Salicylic acid in beaker can be measured. However, Tube
B contains Salicylic acid in presence of protein (0.5 ml egg albumin) and hence the protein
binding of Salicylic acid takes place. This allows only free Salicylic acid left after protein
binding to equilibrate through semipermeable membrane, which reduces the concentration of
Salicylic acid in beaker.Further, reduction in concentration of Salicylic acid takes place in
tube C because of increased presence of protein (1 ml egg albumin). This increases protein
binding of salicylic acid, thereby reducing free Salicylic acid in the tube and in beaker
too.Salicylic acid present in beaker (non protein compartment) is estimated by adding ferric

Table1. Calibration curve of salicylic acid
Concentration Absorbance
At 547nm
10
20
30
40
50
60

nitrate solution. The reaction of Salicylic acid with ferric nitrate produces an intensely
colored complex, whose maximum absorbance can be detected at 540nm
spectrophotometrically.
The fraction of drug unbound (FU) in plasma is given as
FU = Concentration of unbound drug in plasma
Total plasma drug concentration
PROCEDURE
Calibration Curve Of Salicylic Acid:
 Preparation of colouring agent: Dissolve ferric nitrite (4g) in100ml distilled

water.
 Preparation of standard stock solution: weigh accurately 100 mg of salicylic

acid and dissolve in 100ml of distilled water using 5ml alcohol. Take 10 ml of this
solution and dilute to 100ml with water.
 Preparation of working solution: from stock solution, pipette out 0.2, 0.4, 0.6,
0.8 and 1 ml into 10ml volumetric flask and add 4ml colouring agent. Dilute
resulting solution to 10ml with water to get concentration in range of 2 to
10mcg/ml.
 Measure the absorbance: record absorbance of the working solution of salicylic

acid at ʎmax of 540nm using UV visible spectrophotometer against water as blank.
Plot a graph of absorbance versus concentration.
Protein binding study of drug
 Take three hollow tubes and mark them as A, B and C.

 To one end, tie a semipermeable membrane in such a way that a sack is formed.
 Place 1ml of 0.1%Salicylic acid and 1ml of distilled water in tube A.
 Place 1ml of 0.1%Salicylic acid and 0.5 ml of egg albumin and 0.5 ml distilled water
in tube B.
 Place 1ml of 0.1% Salicylic acid and 1ml of egg albumin in tube C.

OBSERVATIONS
Table 2. Percent cumulative drug release with and without protein through egg
membrane
Percent
cumulative
Time(min) drug release Tube A Tube B Tube c
10
20
30
40
50
60
CALCULATIONS
A.Determination of unknown concentration of Salicylic acid
Determine unknown concentration of Salicylic acid from receptor compartment by using

following equation
Y=mX+c
Where,Y= absorbance,m= slope,X= concentration, c = the intercept.
B.Amount of drug diffused (mg)
Amount of drug diffused =[Concentration ( µ/ml) x (volume of diffusion medium) x

(dilution factor)]/1000
C. Dilution factor
Dilution factor = volume of diluted sample (ml) / volume of sample removed (ml)
D. Percent cumulative drug release
Percent cumulative drug release = (amount of drug diffused x 100) /total amount of
dose

 Place the tubes in a 50 ml of distilled water and pipette out 5 ml of sample from
beaker at the intervals of 15,30, 45, 60, 75 and 90 min.
 Add 4ml of coloring agent and 1ml of distilled water and determine absorbance at 540
nm. Replace the fluid with 5ml of distilled water in the beaker.
 Plot comparative curves of amount of Salicylic acid present in non protein
compartment versus time.
RESULT
The percent release of Salicylic acid without any proteins was found to be _______,
with 0.5 ml of egg albumin was______, and with 1ml of egg albumin was_______.
REFERENCES

pharmacokinetics, first edition, Trinity publishing house, Satara, p.66-70

Exp.No: 10 Date:
IN SITU DRUG ABSORPTION STUDY OF SALICYLIC ACID BY USING

EVERTED CHICKEN ILEUM
AIM
To perform in vitro absorption-permeation study of aspirin tablet .
REQUIREMENTS
Glasswares: Beaker, test tube, funnel, volumetric flask, etc.

Chemicals: Sodium lauryl sulphate, phosphate buffer pH 5, distilled water, Krebs ringer
solution pH 7.4,ferric chloride and Aspirin tablet
Instruments:Weighing balance, dissolution apparatus,UVspectrophotometer
THEORY
Good oral bioavailability occurs when the drug has maximum permeability and
maximum solubility at the site of absorption. The extent of absorption of drug , thus, could be
predicted based on permeability and solubility measurements. Hence, the intestinal
permeability represents one essential part in the prediction of oral bioavailability.The
intestinal permeability data can be used in preformulation studies to evaluate the effects of
various pharmaceutical excipients on drug absorption. A number of in vitro methods for
assessing the intestinal permeability of a given drug have been developed. absorption
(permeability) studies based on isolated intestinal sacs are routinely performed. The
advantages of this model are that it contains all the types of cells and mucus layer; it is
relatively fast and inexpensive; and it can be used for preformulation studies. This kind of
model is suitable for measuring kinetic parameters with high reliability and reproducibility
The chicken small intestine could be a useful model for intestinal absorption based on the
assumption that membrane permeability of drugs is not species-dependant, since the
composition of plasma membrane of intestinal epithelial cells is similar across the species.
Thus the permeability across the chicken intestinal segment could be expected to be the same.
Furthermore, the model would have advantages as less labor intensive, less time consuming,

lower cost per assay and, since slaughtered chicken is used, no special permission from
animal ethical committees would be required
Design of continuous dissolution-absorption system using everted intestine segment
The in vitro continuous dissolution absorption system design is illustrated in Figure 1. The
system consists of USP dissolution apparatus 1 and a side-by-side perfusion apparatus
holding isolated everted intestine segment. In this system, drug dissolution from the slow
release tablet and permeation across everted intestine occurs simultaneously. Use 1000 ml of
distilled water as dissolution medium maintained at 37 ± 0.5 °C. The perfusion apparatus is
consisted of two glass tubes,Aand B, connected together (Figure 1). Tube B is a bent cannula
at its lower end, and tube A, a straight cannula at its lower end. The distance between two
cannula is kept constant. The isolated everted intestinal segment is fixed between the ends of
tubesAand B as shown in the figure 1. The ends of the intestine are tied in position with a
thread. The apparatus is immersed completely into the dissolution vessel.
Figure 1. Design of in vitro continuous dissolution-absorption system
Labels:1. Dissolution flask, 2. Rotating shaft, 3. Dissolution medium, 4. Basket, 5. Tablet, 6.

Oxygen tube, 7.
Tube B, 8.TubeA, 9. Everted intestine, I. Dissolution absorption system, II.Absorption
(perfusion) apparatus.

PROCEDURE
Preparation of solution:
Krebs ringer solution: Prepare the Krebs ringer solution by combining 6.3 g NaCl, 0.35 g
KCl, 0.14 g CaCl , 0.16 gKH PO , 0.15 gMgSO ·7H O, 2.1 gNaHCO , and 5 g glucose in one
liter of distilled water.
Plotting of calibration curve
Transfer accurately weighed 100 mg of Acyclovir in to 100 ml volumetric flask containing
sufficient quantity of phosphate buffer pH 5.0. Finally adjust volume to get stock solution
containing 100 μg/ml Aspirin Withdraw adequate quantities of aliquots from standard stock
solution in 10 ml volumetric flask and dilute with distilled water to get the concentration of 2,
4, 6, 8, 10, 12, 14, 16, 18 and 20 μg/ml of aspirin. Measure the absorbance of these dilute
solutions at a max of 547 nm by using double beam UV spectrophotometer against a blank .
Plot the graph of absorbance versus concentration and determine slope, intercept and
coefficient of correlation.
Isolation of everted intestine
Bring male white Leghorn chicks weighing between 500 and 600 g from the local market.
For isolation of everted intestine, slaughter the chicks, perform a median incision of the
abdomen and free the small intestine. Clean the lumen carefully from mucus by rinsing with a
pH 7.4 buffer solutions (Krebs ringer solution).Remove an intestinal segment of the first 6 cm
length and transfer to oxygenated Krebs ringer solution. Wash it thoroughly with warm Krebs
ringer solution. Turn back the proximal extremity of the intestine and ligate on a glass rod to
form an everted bag. Alternatively, chick intestine can be obtained from slaughter house by
taking proper care of providing oxygenated Krebs ringer solution to intestinal segment
removed. This will eliminate the requirement of taking permission of Ethics Committee for
performing this experiment.
Dissolution absorption studies
1.Maintain the dissolution medium consisting of 900 ml of phosphate buffer pH 5.0 at 37 ±

0.5 °C. Clamp a fresh intestinal segment to perfusion apparatus, as shown in the figure 1
2. Fill Krebs ringer solution in perfusion apparatus and record total volume of absorption
compartment (tube A and tube B of perfusion apparatus).

3. Place the perfusion apparatus into dissolution medium and aerate it.
OBSERVATION
Table 1. Percent cumulative drug release(%CDR)

Time(min) Absorbance Concentration Cumulative CADD/unit
At 537nm amount of drug area
release (mg)
10
15
20
25
30
Table 2.Amount of Aspirin diffused

diffused (mg)
10
20
30
40
50
60

4. Place aspirin tablet into basket and rotate it at 50 rpm.
5. Withdraw 2 ml sample at interval of 10 min up to 120 min and determine released aspirin
spectrophotometrically at 537 nm.
6.Withdraw the transported drug from absorption compartment with replacement of Krebs
ringer solution at 13 min to 123 min at the interval of 10 min and analyze it
spectrophotometrically for transported Aspirin at 537 nm. To allow time for drug to circulate
from the dissolution vessel to the everted intestine surface, absorption samples should be
collected 3 min later than their corresponding dissolution samples.
7. Repeat whole experiment in triplicate (n=3) using fresh dissolution medium as well as
fresh intestinal segment each time.
8. Plot the graph of percent drug released versus time.
9. Plot the graph of cumulative amount of drug diffused versus time and determine slope of
linear portion as steady state appearance rate (mg/min) namely the amount of a compound
traversing the tissue in time t (min).

CALCULATIONS:
1. Concentration of diffused drug ( g/ml)
Plot the graph of absorbance versus concentration and determine slope and intercept.
Y=mX+ c
Where,Y=absorbance,m=slope,X=concentration ( g/ml), c = intercept.
2. Cumulative amount of drug diffused (mg)

Cumulative amount of drug diffused (CADD) =
[Concentration ( g/ml) x (volume of diffusion medium) x (dilution factor)]/1000
Surface area (A) of chicken intestine (cm )
A=2 rl
Where, r = internal radius of everted intestine, l = length of everted intestine.
4. Dilution factor
Dilution factor = volume of diluted sample (ml)/ volume of sample removed (ml)
5.Amount of drug released in dissolution medium (mg)
Amount of drug released =
[Concentration ( g/ml) x (volume of dissolution medium) x (dilution factor)]/1000
6. Percent cumulative drug release
Percent cumulative drug release = (Amount of drug released) x (100 /strength of tablet)
7.Apparent permeability (cm/s)
Where, dQ/dt = steady state appearance rate, namely the amount of a compound traversing
the tissue in
time t (min), A = exposed area of the tissue (cm ), C = initial concentration of the drug in the
donor
compartment.

REPORT
1.Percent drug release of marketed aspirin tablet in phosphate buffer is _________%in 2 hr.
2.Amount of Aspirin diffused through intestinal membrane is _________ in 2hr
REFERENCES
pharmacokinetics, first edition, Trinity publishing house, Satara, page no:41-46

CALCULATIONS;
1. Determination of concentration of diffused drug ( g/ml)
Plot the graph of absorbance versus concentration and determine slope and intercept.
Y=mX+ c
Where,Y= absorbance,m= slope,X= concentration ( g/ml), c = intercept.
2. Cumulative amount of drug diffused (mg)
Cumulative amount of drug diffused (CADD) =
[Concentration ( g/ml) x (volume of diffusion medium) x (dilution factor)]/1000
3. Surface area (A) of cellophane membrane
A= r
Where, r = radius of cellophane membrane exposed to diffusion medium.
4. Cumulative amount of drug diffused per unit area (CADD/cm )
CADD/cm = CADD/Area of cellophane membrane
5. Dilution factor
Dilution factor = volume of diluted sample (ml)/ volume of sample removed (ml)
6. Flux (J )
Slope (J ) of linear portion of plot of amount of drug diffused per unit area versus time.
7. Permeability coefficient (K )
KP = Jss/c
Where,C = initial concentration of the drug in donor compartment (mg)5

Exp.No: 11 Date :
DIFFUSION STUDY OF SALICYLIC ACID BY USING EGG MEMBRANE
AIM
To study the in vitro permeation of paracetamol.
REQUIREMENTS
Paracetamol solution (100µg/ml), egg membrane, 100ml phosphate buffer of pH 5.8,

magnetic stirrer.
PRINCIPLE
The in vitro permeation of drug can be studied by open end tube with one end tied
with egg membrane. The open end tube is made of glass with a contact area of 3.8cm2. The
drug solution is taken in a open end tube which is immersed in a beaker containing phosphate
buffer of pH 5.8.The solution in beaker is continuously stirred by means of a magnetic stirrer
. Solution samples, 10 ml aliquots, are withdrawn from the beaker at various time intervals.
The beaker is refilled with phosphate buffer of pH 7.8 to keep the volume constant during the
experiment.
PROCEDURE
Calibration curve of Paracetamol in phosphate buffer of pH 5.8
 Transfer 100mg of paracetamol to 100ml standard flask and make up the volume with
phosphate buffer of pH 5.8.
 Take 1ml of prepared solution and make up to 100ml.
 Take 10ml of above solution and make up to 100ml.
 Then take 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and make up to 10ml and the
absorbance was noted at 243nm UV Spectrophotometrically.

Table1. Calibration curve of paracetamol
Concentration Absorbance
At 243nm
10
20
30
40
50
60
Table 2. Permeation of paracetamol through egg membrane
diffused (mg)
10
20
30
40
50
60

In vitro release and permeation
 Taken an open end tube which is tied with an egg membrane at one end.
 Then add 10.0ml of 100mcg/ml solution of paracetamol to the open end tube.
 It is kept in contact with a 100.0ml phosphate buffer at pH 5.8 and 10 ml of sample

was taken at an interval of 0.5, 1, 1.5, 2 hours respectively.
 The absorbance of the given solution was observed at 243nm spectrophotometrically.
 Then the concentration was determined from the plotted calibration curve.
 The cumulative amount of drug diffused is calculated and then CADD/unit area is
determined.
 A graph is plotted by taking CADD/unit area in y-axis and time in x-axis.
 The slope of the graph will given the flux JSS.
 Then permeability coefficient is determined by dividing flux with initial

concentration.
REPORT
Permeability coefficient and flux of paracetamol through egg membrane was found to be
and respectively.
REFERENCE

pharmacokinetics, first edition, Trinity publishing house, Satara, p.62-65.

Exp.No:12 Date :
FORMULATION AND EVAULATION OF TRANSDERMAL PATCH OF

PARACETAMOL USING HPMC
AIM
To formulate and carry out in vitro diffusion study of transdermal patch of paracetamol.
MATERIALS REQUIRED
Paracetamol, HPMC, glycerin
PRINCIPLE
Transdermal Drug delivery systems are the novel controlled drug delivery devices
where the device is formulated as patch that is applied on to the skin. Transdermal drug
delivery offers a route, which bypasses the hepatic first pass metabolism by providing the
drug in a controlled manner across the skin into the systemic circulation. Transdermal drug
delivery route can be applied to a wide variety of therapeutic categories of drug like Anti-
anginals, Anti-Hypertensives, Anti-inflammatories, proteins and peptide drugs etc. poorly
soluble drugs are represented by low absorption and weak bio-availability. One possibility of
increasing the solubility is the reduction of particle size. However, the fine particle may not
always produce the expected faster dissolution and absorption. These problems require better
technologies in processing new drug delivery system. Solid dispersion of solution method can
be used for these purpose, solid dispersion refers to dispersion of one or more active
ingredients in an inert carrier or matrix at a solid stae. Solid dispersion can be prepared by
melting (fusion) method. The solid dispersion may also be called solid state dispersion. The
term co-precipitate has been used to those preparations obtained by solvent.
PROCEDURE
 The film was prepared by dissolving the 4% Hydroxyl propyl methyl cellulose by
continuous stirring.
 Added 0.5 ml of glycerin into the above solution.
 100mg of Paracetamol was incorporated and stirred thoroughly until the drug is
continuously dissolved.
 Removed the air bubbles by ultra sonification.
 Pour the above mixture into the mould.
 Dried at room temperature for 24hours.
EVALUATION
A patch of 1cm2 area was taken and extracted with 5ml of distilled water and 1ml of the
above solution was taken and absorbance was measured at 257nm and the amount of drug
present in 1cm2 area of patch was calculated.
PHYSICAL EVALUATION
Percentage moisture loss
The films were weighed accurately and kept in a desiccators containing anhydrous
calcium chloride. After 3days, the films were taken out and weighed. The moisture loss
was calculated using the formula:
% moisture loss =final weight – initial weight × 100

Initial weight
Folding endurance
The folding endurance was measured manually for the prepared films. A strip of films
(3×4 cm) was repeatedly folded at the same place till it broke. The number of times the
film could be folded at the same place without breaking/cracking gave the value of
folding endurance.
Mass variation
The patches were subjected to mass variation by individually weighing 10 randomly

selected patches. Such determinations were carried out for each formulation.
RELEASE STUDIES
In vitro diffusion study
 In vitro diffusion study was carried out by using Franz- diffusion cell. In this method
egg membrane is used as model membrane.
 The membrane was placed between the donor compartment and the reservoir
compartment (phosphate buffer pH 5.8).
 The patch was placed on the membrane and the compartments clamped together.
 The receptor compartment (70ml capacity) was filled with phosphate buffer of pH 5.8
and hydrodynamics in the receptor compartment was maintained by stirring with a
magnetic bead at 100rpm.
 5ml of sample is withdrawn and replaced with receptor medium.
 Amount of drug present is assayed spectrophotometrically at 243nm and amount of
drug release at various time intervals was calculated.
REPORT
REFERENCE
1.Pravin Gavali et al Design and development of hydroxypropyl methylcellulose (HPMC)

based polymeric film of Enalapril maleate Int.J. PharmTech Res.2010,2(1):264-284

Exp.No: 13 Date :
DISSOLUTION STUDY OF UNCOATED MARKED TABLETS
AIM
To carry out dissolution study of uncoated marketed tablets of paracetamol.
REQUIREMENTS: Paracetamol tabets, rotating paddle type apparatus. Phosphate buffer PH

5.8,UV- Vis spectrophotometer
PREPARATION OF REAGENT:
Phosphate buffer PH 5.8: Take 50ml of 0.2 M potassium di hydrogen phosphate in

200ml volumetric flask to this add 3.6 ml of 0.2 M NaOH and make up to the volume with
distilled water.
PRINCIPLE:
Dissolution is a process in which a solid substance solubilize in a given solvent i.e.,

mass transfer from the solid surface to the liquid phase. Its rate is defines as the amount of
solid substance that goes into solution per unit time under standard condition of temperature,
PH, solvent composition and constant solid surface area.
Noyes and Whitney (1897) and other investigators studied the rate of dissolution of
solid drugs. According to their observations, the steps in dissolution include the process of
drug dissolution at the surface of the solid particle, thus forming a saturated solution around
the particle. The dissolved drug in the saturated solution, known as the stagnant layer,
diffuses to the bulk of the solvent from regions of high drug concentration to regions of low
drug concentration.
According to modified Noyes-Whitney equation
dc/dt = (DA ko/w (Cs –Cb)) / V h
Where, dc/dt = rate of dissolution.Cs-Cb = concentration gradient.

V = volume of distribution. h = thickness of stagnant layer.
Ko/w = partition coefficient. D = diffusion coefficient.
A = surface area of solid.

The Noyes–Whitney equation shows that dissolution in a flask may be influenced by the
physicochemical characteristics of the drug, the formulation, and the solvent. Drug in the
body, particularly in the gastrointestinal tract, is considered to be dissolving in an aqueous
environment. Permeation of drug across the gut wall (a model lipid membrane) is affected by
the ability of the drug to diffuse (D) and to partition between the lipid membranes. A
favourable partition coefficient (K oil/water) will facilitate drug absorption.
In addition to these factors, the temperature of the medium and the agitation rate also
affect the rate of drug dissolution. In vivo, the temperature is maintained at a constant 37°C,
and the agitation (primarily peristaltic movements in the gastrointestinal tract) is reasonably
constant. In contrast, in-vitro studies of dissolution kinetics require maintenance of constant
temperature and agitation. Temperature is generally kept at 37°C, and the agitation or stirring
rate is held to a specified rpm (revolutions per minute). An increase in temperature will
increase the kinetic energy of the molecules and increase the diffusion constant, D. Moreover,
an increase in agitation of the solvent medium will reduce the thickness, h, of the stagnant
layer, allowing for more rapid drug dissolution.
Factors that affect drug dissolution of a solid oral dosage form include (1) the physical
and chemical nature of the active drug substance, (2) the nature of the excipients, and (3) the
method of manufacture.
The best available tool today which can at least quantitatively assure about the
biologic availability of a drug from its formulation is its in vitro dissolution test. For an in
vitro test to be useful, it must predict the in vivo behaviour to such an extent that in vivo
bioavailability test need not be performed. The efforts are mainly aimed at mimicking the
environment offered by the biological system. The dissolution apparatus has evolved
gradually and considerably from a simple beaker type to a highly versatile and fully
automated instrument. Mainly two types of dissolution apparatus are there, rotating basket
and rotating paddle type apparatus.

PROCEDURE:
 Carry out the dissolution test for tablets using 900 ml of phosphate buffer P H 5.8 as
medium.
 Rotating the paddle at 50 rpm for 30 minutes withdrawn a suitable volume (1ml) of
the sample.
 Filter promptly through a membrane filter disc with an average pore diameter not
greater than 1 micrometre.
 Reject the first few ml of the filtrate and dilute a suitable volume of the filtrate with
the same solvent.
 Measure the absorbance of the resulting solution at the maximum at about 243nm.
REPORT:
The percentage drug release from given tablet after 30min was found to be
REFERENCES
USP30–NF25 Page 1269 Pharmacopeial Forum : Volume No. 27(3) Page 2495

Biopharmaceutics and Pharmacokientics A Pracitcal Manual Compiled by Deepa MK

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Biopharmaceutics

Compiled By: Deepa MK,M.Pharm.,

Sl . Date Experiments Page No. Marks Sig.

3 Data Analysis III

8 Demonstration Of In Vitro One Compartment

9 Studies On Protein Binding Of Salicylic Acid

10 Diffusion Study Of Salicylic Acid By Using Egg

11 In Situ Drug Absorption Study Of aspirin tablet By

12 Formulation And Evaluation Of Transdermal Patch

13 Dissolution Study of Uncoated Marked Tablets

Time Plasma Plasma

Laboratory Manual of Biopharmaceutics and Pharmacokinetics 1

To determine the pharmacokinetic parameters such as Cmax, tmax, KE, , t ½, ,

Laboratory Manual of Biopharmaceutics and Pharmacokinetics 2

3. Elimination rate ( KE):Elimination rate constant is calculated from the terminal

4. Biological Half-life ( t 1/2)

2. The area under curve from t* to infinity by using formulae

t* = time at which blood sampling is stop

Laboratory Manual of Biopharmaceutics and Pharmacokinetics 4

Laboratory Manual of Biopharmaceutics and Pharmacokinetics 6

1.P L Madan. Biopharmaceutics and pharmacokinetics.1st edition; p.196-197

2. C.Vijayaraghavan ,Experimental biopharmaceutics and pharmacokinetics ,1st edition. P.61-

Laboratory Manual of Biopharmaceutics and Pharmacokinetics 8

To determine the pharmacokinetics parameters like KE , Vd,and C0 following 250mg

In one compartment model, the body is considered as single, kinetically homogenous

Laboratory Manual of Biopharmaceutics and Pharmacokinetics 10

Blood and other

The general expression for rate of drug presentation to the body is

= Rate in (availability) – Rate out (elimination)------------------------ (1)

Since rate in or absorption is absent, the equation becomes;

= -Rate out ------------------------------------------------------------------(2)

If the rate out or elimination follows first-order kinetics, then:

Elimination rate constant (KE)

Laboratory Manual of Biopharmaceutics and Pharmacokinetics 12

It is the measure of extent of distribution of drug and is expressed in litre.

Where X 0 is dose administered, C0 is initial plasma concentration.

Elimination rate constant (KE) =

Volume of distribution (Vd) =

Elimination half-life (t1/2) =

Initial concentration (C0) =

1.D.M Brahmankar, Sunil B. Jaiswal, Biopharmaceutics and Pharmacokinetics A Treatise,

Laboratory Manual of Biopharmaceutics and Pharmacokinetics 14

Laboratory Manual of Biopharmaceutics and Pharmacokinetics 16

Laboratory Manual of Biopharmaceutics and Pharmacokinetics 17

, a hybride constant ,then

exponential approaches zero whereas, first exponential retains some finite

At this time equation (2 )reduces to,

In log form above equation is

Laboratory Manual of Biopharmaceutics and Pharmacokinetics 18

Laboratory Manual of Biopharmaceutics and Pharmacokinetics 20

Laboratory Manual of Biopharmaceutics and Pharmacokinetics 22

Plasma Extrapolated Residual

Laboratory Manual of Biopharmaceutics and Pharmacokinetics 23

Absorption rate constant :

Elimination rate constant:

1.S.B.Bhise, R.J.Dias et.al, Laboratory manual of biopharmaceutics and

Laboratory Manual of Biopharmaceutics and Pharmacokinetics 24

Time of urine Volume of Concentration

1 0-2 140 250

2 2-4 150 100

Laboratory Manual of Biopharmaceutics and Pharmacokinetics 26

Time of Volume Concentrati Urine midpoint amount excretio

1 0-2 140 250