LAB REPORT

Submitted by Submitted to

Rabea Halim
Kazi Fatema Rahman
ID: 18146006
Lecturer
Section: 01
Department of Pharmacy
Course Name: Pharmacology III Lab Brac University
Course Code: PHR416

Session: Summer 2021

Date of submission: 24.11.21

 INDEX:

Experi
SI Page
ment Name of the Experiment Date:
NO. No.
no.

Determination of Salicylic acid level in

1 1 human blood after oral administration of 22.11.21
Aspirin tablet

Determination of serum cholesterol level by

2 2 22.11.21
Enzymatic method
Experiment 1: Determination of Salicylic acid level in human blood after
oral administration of Aspirin tablet
Principle:
Aspirin is a non-steroidal anti-inflammatory drug used as an analgesic to relieve minor aches and
pains, as an antipyretic to reduce fever, as an anti-inflammatory medication and as anti-platelet
aggregation agent used to prevent heart attacks or strokes due to clots.
Mechanism of action of Aspirin:

Tissue injury

Phospholipids in cell membrane

Phospholipase A2

Aspirin inhibits Arachidonic acid Aspirin inhibits

COX-1 COX-2

Constitutive Prostaglandin Inflammatory Prostaglandin

-GI mucosa protection -Recruit inflammatory cells

-Aid platelet aggregation -Sensitize pain receptors

-Regulate
hypothalamic
temperature control
Toxicity of aspirin:

1. Acute toxicity: Acute over dosage of aspirin can cause nausea, vomiting, dehydration,
hyperpnoea, oliguria, and tinnitus. Severe poisoning can cause coma, convulsions,
severe hyperpnoea, and metabolic acidosis.
2. Chronic toxicity: Chronic salicylate poisoning (salicylism) occurs most commonly in
seniors who regularly take large doses of aspirin, often for osteoarthritis, and then
gradually increase their doses or develop renal insufficiency. Signs and symptoms of
salicylism include fever, vomiting, and tachypnea.
3. Gastrointestinal toxicity: Frequent intake of aspirin inhibits the formation of
prostaglandins, in turn depleting the protective barrier in the stomach and leading to
peptic ulcers.
Significance of determination of salicylic acid level in blood:

The therapeutic range of salicylate is 15-30 mg/dL. Patients are often symptomatic at salicylate
concentrations higher than 40-50 mg/dL. Patients with salicylate concentrations approaching or
exceeding 100 mg/dL usually have serious or life-threatening toxicity. Patients with chronic
poisoning who have levels of 60 mg/dL or greater often have serious toxicity.
This experiment is conducted for the determination of aspirin in blood sample by
spectrophotometric method which is based on the formation of a color complex of the drug in
serum with ferric-mercuric reagent. Ferric mercuric is used as reagent to precipitate the plasma
protein in human serum. The absorbance of the colored complex is then measured at 540nm for
maximum absorption.
Chemical structure of Aspirin:
Apparatus:
1. Test tubes
2. Volumetric flask
3. Centrifuge machine
4. Micropipette
5. Electronic balance
6. UV Spectrophotometer
7. Syringe

Reagents:
1. Aspirin tablet
2. Mercuric chloride
3. Ferric nitrate
4. Hydrochloric acid
5. Distilled water

Procedure:

Preparation of 250ml of 1N HCl:

20.6ml of concentrated HCl was dissolved in distilled water and volume was made up to 250ml
with distilled water.

Preparation of 250ml of Ferric Mercuric reagent:

10gm of Mercuric chloride was dissolved in 212.5ml distilled water followed by addition of
30ml HCl and 10gm of Ferric nitrate and the volume was made up to 250ml with distilled water.

Reagents Quantity Quantity Quantity Quantity

Mercuric chloride 40gm 20gm 10gm 5
Distilled water 850ml 425ml 212.5ml 106.25ml
HCl 120ml 60ml 30ml 15ml
Ferric nitrate 40gm 20gm 10gm 5gm
Final volume 1000ml 500ml 250ml 125ml
 Preparation of Standard curve:

1. 10 mg of Salicylic acid was dissolved in 100ml distilled water resulting 0.1mg/ml

stock solution.
2. From this 100ml solution, 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml and 1ml solution was withdrawn
followed by addition of 9.8 ml, 9.6 ml, 9.4 ml, 9.2 ml and 9 ml distilled water respectively to
form 10 ml solution which resulted in 2 µg/ml, 4 µg/ml, 6 µg/ml, 8 µg/ml and 10 µg/ml
standard solution of the drug respectively.
3. A blank containing 10 ml distilled water was also taken.
4. 5 ml Ferric-Mercuric reagent was added to each of the 6 test tubes.
5. The absorbance for each concentration of the corresponding volumes was measured at
λmax= 540 nm.
6. A graph of Absorbance versus concentration was plotted to obtain the standard curve of
Aspirin.

Data table:

Concentration (µg/ml) Volume (ml) Absorbance (λmax=

540nm)
2 0.2 0.043

4 0.4 0.062

6 0.6 0.080

8 0.8 0.101

10 1.0 0.122
 Standard curve of Aspirin
0.14

0.12
f(x) = 0.01 x + 0.02
0.1 R² = 1
Absorbance (AU)

0.08

0.06

0.04

0.02

0
1 2 3 4 5 6 7 8 9 10
Concentration (µg/ml)

Preparation of Blood Sample:

1. After 1hr of the administration of 75 mg Aspirin tablet, blood was withdrawn from the
subject and 5ml of the blood was centrifuged for 20 minutes.
2. The centrifugation of the blood sample resulted in three distinct layers of serum, plasma
and blood cells. 1ml of supernatant serum was withdrawn and transferred to a test tube
followed by addition of 5ml Ferric-Mercuric reagent.
3. The absorbance was measured at λmax= 540nm.

Data table:

Sample Absorbance (λmax=

540nm)
Serum (male) 0.565

Preparation of Blank solution:

1. 5ml of Ferric Mercuric reagent was added in 10 ml distilled water.

2. The absorbance was measured at λmax= 540 nm.
Calculation:

Equation from standard curve: y= 0.0098x+0.0225

Absorbance of serum sample= y= 0.565, x=Concentration of salicylic acid in
serum:
0.565= 0.0098x+0.0225
Therefore, x= 55.36 µg/ml

Result:
The concentration of the salicylic acid present in the serum of volunteer was found to be
55.36µg/ml.

Discussion:
Aspirin is hydrolyzed to salicylic acid in the stomach as soon as it enters into the blood
circulation. Aspirin in the standard solution also hydrolyzed to salicylic acid that formed complex
with the ferric-mercuric reagent. This complex gave a purple color that was determined for
salicylate content in spectrophotometer in the visible range. Similarly, the reagent also formed
complex with the salicylate in the blood serum and the concentration of salicylic acid in subject’s
(male) blood after 1hr of administration of 75mg Aspirin tablet was found to be 55.36 µg/ml. The
normal therapeutic range of salicylic acid lies within 150-300 µg/ml (15-30 mg/dL) which is
obtained during 4-6 hours of administration of aspirin but since blood concentration was taken 1
hour after administration, the concentration of salicylic acid in subject’s blood was much below
the therapeutic value. Moreover, due to difference in drug metabolism pattern of individuals,
hydrolysis of aspirin to salicylic acid may not be sufficient enough to reach the therapeutic range.
Precaution:
1. All the apparatus and reagents were handled carefully.
2. Measurements were done properly.
Experiment 2: Determination of serum cholesterol level by Enzymatic method
Introduction:
Cholesterol is a steroidal compound which is one of the most important constituents of the human
body. It serves several important functions such as-
i. It is a structural component of cell membranes. It provides fluidity to the membrane.
ii. Precursor for the synthesis of all steroidal compounds in the body including corticosteroids,
sex hormones, bile acids.
iii. Precursor of vitamin D
iv. Precursor for synthesis of cholic acid (bile acid) which aids in fat digestion.
The biosynthesis of cholesterol takes place in the liver, involving five major steps-
i. Acetyl-CoA is converted to 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA).
ii. HMG-CoA is converted to mevalonate.
iii. Mevalonate is converted to isopentenyl pyrophosphate (IPP).
iv. IPP is converted to squalene.
v. Squalene is converted to cholesterol.
Principle:
Cholesterol level is determined by enzymatic hydrolysis and oxidation. Cholesterol ester is
hydrolyzed to free cholesterol using the enzyme cholesterol esterase. Then free cholesterol, in the
presence of oxygen, is converted to Cholesten-3-one and H 2O2 with the help of the cholesterol
oxidase enzyme. The H2O2 reacts with 4-amino phenazone and phenol in the presence of
peroxidase enzyme, forming 4(p-benzoquinone) mono iminophenazide, which is pink in color.
Absorbance of pink cholesterol at 546 nm is directly proportional to the amount of cholesterol
present.
Reaction:
• Cholesterol ester + H2O ----cholesterol esterase Cholesterol + fatty acids
• Cholesterol + O2 ---cholesterol oxidaseCholesten-3-one + H2O2
• 2H2O2+ Phenol + 4- amino phenazone----peroxidase--Iminophenazide dye (pink) + 4H2O
Chemical structure of cholesterol:
Blood cholesterol level:
Blood Cholesterol Normal (mg/dL) Borderline (mg/dL) High risk (mg/dL)
Total cholesterol <200 200-240 >240
Low density <130 130-160 >160
lipoprotein (LDL)
High density >60 35-60 <35
lipoprotein (HDL)

Reagents Used:
a. Composition of the enzyme reagent:
4×30ml, 3×250ml or 4×100ml enzyme reagent comes as a kit
i. Phosphate buffer (pH 6.5) 100mmol/L
ii. 4-amino phenazone 0.25mmol/L
iii. Phenol 5mmol/L
iv. Peroxidase >5KU/L
v. Cholesterol esterase >150U/L
vi. Cholesterol oxidase >100U/L
vii. Sodium azide (preservative) 0.05U/L
b. 3ml cholesterol standard solution of 200 mg/dL or 5018 mmol/L
Storage condition of reagent:
• Needs to be stored away from light in a dark place.
• Storage temperature: 2-8 °C

Apparatus:
i. Test tubes
ii. Micropipette
iii. Centrifuge machine
iv. UV spectrophotometer
v. Syringe

Procedure:
1. 3 test tubes were taken labeled as: a) Standard b) Blood c) Blank
2. 10 μL of provided standard cholesterol was taken in test tube (a).
3. Blood was collected by syringe in a small glass tube shaken well to mix anticoagulant.
4. The blood was then transferred into centrifuge tube and centrifuged for 15 minutes at a high
speed to separate the protein to obtain the serum.
5. 10 μL of serum was taken in test tube (b) with micropipette.
6. 3 ml enzymatic reagent was added in each of the three test tubes.
7. All the test tubes were kept in a dark place for about 10-15 minutes.
8. The absorbance for standard, serum and blank was measured at λmax= 500 nm.
Calculation:
Sample Absorbance (λmax= 500 nm)
Blank 0.0
Standard cholesterol 0.188
Serum sample 0.160

Concentration of cholesterol in blood= (Absorbance of serum sample × Concentration of standard

cholesterol)/Absorbance of standard cholesterol
= (0.160 x 200)/0.188
= 170 mg/dL
Result:
The blood cholesterol level of the volunteer was found to be 170 mg/dL.
Comment:
The blood cholesterol level was found to be 170 mg/dL. The normal range of blood cholesterol is
<200 mg/dL. So, the blood cholesterol level of the volunteer was within the normal range.
Experimental precautions:
1. All the apparatus was washed and handled carefully.
2. Micro pipettes were handled carefully while performing the task specially during taking the
reagents.
3. All the three test tubes were kept in a dark place and covered with foil paper properly before
taking the absorbance.
4. Tips for the micropipette were used for taking each individual substance and disposed
immediately after use.
5. All the chemical reagents were measured accurately.
6. Cuvette was cleaned properly before measuring the absorbance.