TOPIC:- ANALYSIS OF PURITY OF DRUGS

SUPERVISED BY :- SUBMITTED BY:-

[Link] GAURAV
CHOPRA
[Link](H) BIOTECH.
4TH YEAR/8TH SEM.
7440070026
• This project includes the analysis of drugs related to their purity
through various techniques which check different parameters of the
LEVOFLOXACIN drug and compare them with reference drug.

• With the help of chromatographic techniques the sample drug’s results

are compared with that of reference standard drugs by applying tests
given in pharmacopia, which constitute procedures for analysis of
drugs and excipients.
• In pharmaceutical industry there are many parameters related to purity
of drugs which need to be tested. These parameters are checked by
different techniques like dissolution apparatus to check dissolution of
the drugs, uniformity of content in the drugs by HPLC, disintegration
of drugs, moisture content of drugs by karlfischer reagent, sulphate ash
testing etc.

• The most important analytical technique used during the various steps
of drug development and manufacturing is the separation technique:
High Performance Liquid Chromatography (HPLC).
 Levofloxacin is a synthetic chemotherapeutic antibiotic
drug .
 The empirical formula is C18H20FN3O4 ,
Molecular weight is 361.368 g/mol
 It is slightly soluble in water, soluble in glacial acetic acid,
slightly soluble or soluble in dichloromethane, slightly soluble
in methanol.
 It is Antibacterial.
 Levofloxacin should be kept in a tightly closed container, protected
from light
Levofloxacin is limited to the treatment of proven serious and lifethreatening
bacterial infections such as:

Urinary Tract Infections

Skin infections

Inhalational Anthrax

Acute Pyelonephritis
There are 3 sections of testing the drug:

Raw material (RM)

Packaging material(PM)

Finished goods (FG)

 RAW MATERIAL: Levofloxacin hemihydrate

TESTS ARE:

 KARLFISCHER METHOD

 SULPHATED ASH TEST

These test are performed to detect the impurities and moisture

content present in raw material of drug
 Karl Fischer titration is a classic titration method in analytical
chemistry that uses volumetric titration to determine trace amounts of
water in a sample.
 Karl Fischer method uses Karl Fischer reagent, which reacts
quantitatively and selectively with water, to measure moisture
content. Karl Fischer reagent consists of iodine, sulfur dioxide,
a base and a solvent, such as alcohol.
formula used: T.V X k.f f *1oo/(sample weight)*100
According to I.P. :
for Levofloxacin water content limit should NMT 3 percent.
titter vol =5.7003mg/ml
karlfisher factor = 0.974ml
sample weight: 0.2061gm
By applying the given formula:
The result comes out to be 0.0269 or 2.69percent
 This test is usually used for determining the content of inorganic
impurities in an organic substance.

Procedure
This test is performed in a muffel furnace.

Firstly we set the temperature at 800 ±50 degrees for 30 minutes. Then
we keep the empty crucible in the mussel fuurnace until it becomes
red hot. After that we cooled it in a dessicator

Then we measure the weight of empty crucible . Then we take 1 gm of

levofloxacin hemihydrate sample and we moisten the sample with
small amount of 1m sulphuric acid
Then we again put this into the muffel furnace.

After charing for 3 hours we take it out from the furnace and cooled it into
dessicator.

Then again we measure it’s weight. After that we calculate the result and find
how much impurity is present in the sample
Calculation:
 Weight of empty crucible (x) =31.5164g
 Weight of sample (y)=1.0010g
After charing for 3 hours

 Weight of crucible+ residue (z)=31.5169g

 Formula used: (z-x)x100/y

 Result: (31.5169-31.5164)x100/1.0010

=0.05%
 Limit not more than 0.2% according to I.P.

 This means sample is accurate or of good quality.

 After the raw material is passed then it goes to the production area where
the drug is manufactured and it comes in the form of tablet.

 There it is packed.

 Packaging is mainly of two types:

 Strip packing
 Blister packing

 After that it comes to final testing for purification in the quality control lab .
 Now the drug comes to it’s final stage testing.

 Then it goes through purification process.

 The best parameter for the purification process is HPLC which is

commonly known as high pressure liquid chromatography.
 The principle involved is the separation of compounds is due to their
relative differences in travel through the column on application of pressure
exerted through mobile phase or carrying liquid.

 The separation is more effective due to greater surface area achieved due to
very small particle size of stationary phase
 Mobile phase –85 volume of buffer +
15 volume of acetonitrile.

 HPLC Pump(s)

 Sample injector – Injection volume

is 10 ul

 Column or stationary phase –C18

 Detector –wavelength set at 293nm

 Mobile phase waste container

 Gradient
Controller
•
Pump Column
Detector
Injector
Mobile Phases
• AREA(TEST)/AREA(STD)*WT. OF STD/100* DILUTION
*100/[Link] TEST *DIL.*AV. WT. OF TABLETS*1000
RESULT :
By applying formula
results comes out to be
  = 98%
LIMIT ACC TO I.P 95% TO 110%

Thus the sample is passed

• Tablets are solid dosage forms each containing a unit dose of
one or more medicament.

• They are intended for oral administration.

• Some tablets are swallowed whole or after being chewed, some

are dissolved or dispersed in water before administration and
some are retained in the mouth where the active ingredient is
liberated
• Uncoated Tablets:
• Coated Tablets
• Enteric-coated Tablets
• film coated tablets
• sugar coated tablets
• Dispersible Tablets
• Modified-release Tablets
• Soluble Tablets
• Effervescent Tablets
DISINTEGRATION TEST FOR TABLETS

FRIABILITY TEST

DISSOLUTION TEST

UV SPECTROPHOTOMETRY
• This test determines whether tablets or capsules disintegrate
within a prescribed time when placed in a liquid medium under
the prescribed experimental conditions.
• Introduce one tablet or capsule into each tube.
• Add a disc to each tube.
• Suspend the assembly in the beaker containing the specified
liquid and operate the apparatus for the specified time.
• Remove the assembly from the liquid. The tablets or capsules
pass the test if all of them have disintegrated.
• If 1 or 2 tablets or capsules fail to disintegrate, repeat the test
on 12 additional tablets or capsules; not less than 16 of the total
of 18 tablets or capsules tested disintegrate.
• Uncoated Tablets = NMT 15 min
• Film Coated Tablets = NMT ½ hr

BEFORE COATING
• Time taken by 6 tablets to disintegrate = 13min23sec
• Limit – NMT 15min
• AFTER COATING
• Time taken by 6 tablets(Film coated) to disintegrate = 51min16sec
• Limit – NMT 1/2hr
• This means the tablet has passed the DT Test.
 
• . The friability test is designed to evaluate the ability of the
tablet to withstand abrasion in packaging, handling and
shipping.
• It is usually measured by the use of the Roche friabiliator.
• It is used to determine the durability and abrasiveness of
tablets

• FRIABILITY APPARATUS
•Take a sample of 20 tablets.

• Weigh them and calculate the average weight that will be

our initial weight.

•After that put those tablets in to the friability test apparatus.

•Then set the time 4min and rotate the drum 100 times.

•After that remove the tablets and after that measure the weight
again which will be our final weight.

•After then we will apply the given formula:

Friability = Initial wt. - final weight/Initialweight*100

• % Friability = weight of tablets before friability(A) –
weight of tablets after friability (B)*100)/weight of tablets
before friability(A).

• Total weight of 20 tablets =15.66gm

• Friability (before coating)

• Initial weight of 20 tablets=15.660 gm

• After friability weight of 20 tablts= 15.600 gm
• Now % friability = initial wt – final wt*100/initial wt
= 15.660- 15.600/15.660*100
= 0.38 % (NMT 1%)
• The dissolution test determines the cumulative amount of
drug that goes into solution as a function of time”.

Dissolution is a standardised method for measuring the

rate of drug release from a dosage form.
The process consists of two consecutive steps:

1. Liberation of the solute or drug form a dosage form.

2. Solubilisation of the drug particles.

The overall rate of dissolution depends upon the slower of

these two steps.
Introduce the stated volume of the dissolution medium, free from
dissolved air, into the vessel of the apparatus.

Warm the dissolution medium to between 36.5 and 37.5

For each of the tablet or capsule tested, calculate the amount of

dissolved active ingredient in solution as a percentage of the stated
amount where two or more tablets or capsules are placed together

Determine for each test the amount of active ingredient in solution per
tablet or capsules and calculate as a percentage of the stated amount. If
the results do not conform to the requirements at stage S1 given in the
accompanying acceptance table (Table 1), continue testing with
additional tablets or capsules through stages S2 and S3 unless the result
conform at stage S2.
 Stage   Number Tested Acceptance

S1  6 Each unit is not less than D*

+ 5%

Average of 12 units (S1 +S2)

6
S2  is equal to or greater than D,
and no unit is less than  D
-15%.

Average of 24 units
(S1+S2+S3), Is equal t or
greater than D, not, More than
12 2 units are less than D - 15%
S3 
and no unit is less than D -
25%
MEDIUM 54.4 gm of potassium dihydrogen ortho phosphate in 8000
ml of water, pH 5.8

APPARATUS USP TYPE 2 (PADDLE)

RPM (rotation per minute) 50 rpm

TEMPERATURE: 37 +/- 0.4 OC

TIME 30 min
• Acc to I.P.
• Levofloxacin dissolution:
• RPM = 50
• Temperature = 37.4 degree Celsius
• Buffer = 5.8 pH
•  
• 54.4 gm potassium dihydrogen orthophosphate in 8000 ml water
• Dilution: 11mcg
• Avg wt of 20 tablets = 15.66gm
• Avg wt of 1 tablet =783 mg
•  
• Wt of 1st tablet(T1) = 0.7842gm
• Wt of 2nd tablet(T2) =0.7831 gm
• Wt of 3rd table (T3)t= 0.7821 gm
• Wt of 4th tablet (T4) = 0.7832 gm
• Wt of 5th tablet (T5)= 0.7831gm
• Wt of 6th tablet (T6) =0.7821 gm
•
• Wt of standard = 0.1112gm
•  
• Absorbance of standard= 0.620nm
• Absorbance of 1st tablet=0.600nm
• Absorbance of 2nd tablet= 0.601nm
• Absorbance of 3rd tablet= 0.585nm
• Absorbance of 4th tablet=0.584nm
• Absorbance of 5th tablet=0.590nm
• Absorbance of 6th tablet = 0.605nm
• FORMULA USED :
•  
• ABS(TEST)/ABS (STD)*WT. OF STD/100* DILUTION *100/[Link] TEST *DIL.*AV.
WT. OF TABLETS*1000
•  
•  
• FOR T1
• =0.600/0.620*0.1012/100*1/100*900/0.7842*50*0.7830*1000
• = 434.5nm
• = 86.8%
• FOR T2
• =0.601/0.620*0.112/100*1/100*900/0.7831*50*0.783*1000
• =441.6nm
• = 88.1%
• FOR T3
• =0.598/0.620*0.112/100*1/100*900/0.7835*50*0.783*1000
• = 433.3nm
• = 87.8%
• FOR T4
•
=0.604/0.620*0.112/100*1/100*900/0.7826*50*0.783*1000
• = 442.3nm
• = 89.5%
• FOR T5
• =
0.602/0.620*0.112/100*1/100*900/0.7815*50*0.783*1000
• = 443.8nm
• = 87.3%
• FOR T6
• =
0.608/0.620*0.112/100*1/100*900/0.7805*50*0.783*1000
• =439.8nm
• = 89.9%.
• HENCE THE SAMPLE IS PASSED.
• A spectrophotometer is employed to measure the amount of
light that a sample absorbs. The instrument operates by passing
a beam of light through a sample and measuring the intensity of
light reaching a detector.

• The beam of light consists of a stream of photons.

• When a photon encounters an analyte molecule, there is a

chance the analyte will absorb the photon. This absorption
reduces the number of photons in the beam of light, thereby
reducing the intensity of the light beam.

• The light source is set to emit 10 photons per second.

• The intensity of the light reaching the detector is less than the
intensity emitted by the light source.
Transmittance,t
It/Io = t

Absorbance,A
log 1/t = a

Lambert – Beer’s Law

A = abC, A = ξbC
b : Sample Path length, C : Sample Concentration,
a : Absorbance Constant
ξ : Molecular Absorbance Constant
Read (absorbance) Std = 0.470nm
Read (absorbance) test = 0.460nm
Wt. of Std = 0.1012gm
Wt. of Test = 0.1567gm
Av. Weight of tablet = 0.783mg
Dilution = 1000
We know
Reading(Test)/Reading(Std)*Wt. of
Std/100*Dilution*100/Wt. of Test* Av. Weight of tablet
0.460/0.470 * 0.1012/100 * 100/0.1567 * 1000 * 0.783
= 494.9nm
=98.9%
 
Controlling and minimizing the side effects of drugs are the
key issues in assuring the safety of drug therapy.

Drug analysts play a predominant role in assuring the

quality of bulk drug materials and drug formulations and
this is also closely related to the safety issue.

It is recommended that classical assay methods (including

HPLC) be used in exceptional cases only and the time and
energy thus spared be used for more important impurity-
related issues that directly contribute to the safety of drug
therapy.
• Indian Pharmacopoeia Volume 2
•  The Indian Pharmacopoeia [Link]
• Orally Disintegrating Tablets: A Review
• Jaysukh J Hirani, Dhaval A Rathod, Kantilal R Vadalia
• Smt. [Link] B. Pharmacy College, Rajkot-360110, 2Shri
[Link] College of Pharmacy,Rajkot,Gujarat,India. 
• Coulometric Karl Fischer titration simplifies water content
testing
• Wesley G. Poynter Kam Controls Inc., Houston
• Roy J. Barrios Louisiana Offshore Oil Port Inc., New Orleans )
• Acetaaminophen [Link]. Retrieved January 2011.
• Graham GG, Scott KF (2005). "Mechanism of action of
paracetamol". American journal of therapeutics 12.