Product: STAGE-III

1. SOR Procedure
1) check the equipment status tag and make sure
that the instrument is calibrated.
2) Ensure that the optical elements are clean
and ensure that the proper filter is used.

3) After initialization the instrument

set Specification o.R. option.

4) Weight accuratelv ab,ou! I g of the sample

and transfer it into a 100 ml volumetric
flask, add about 7 ml of mefir;ili^il;fiqk;;;ri;;i;ir,.
riurr and dissorve
sample and make up
!he.volumotoi oqgtry,+ qr;ffi;lii .o%ow/v).Ensure thethat the
temperature of the solution is about ZS.C
i+O.S;C):'^'*""'.'
5)
*i.},"'g?if 'bil,li1gh",#llffi:tL5SteXglt*#sethetemperaturesensorand
6) Discard the methanol in the polarimeter
tube and fill it with sample solution. while
filling, take care'that air bubbles are not entrapped and
tighten the end cap
properly.

7) Enter the concentration of sampre, sample ID etc. And observed

the specific
optical rotation or the sample.

t) hardcopy containing five consecutive specific

T*,!E:.a optical rotation readings and

Concentration of sample :
100 x 100

Where LOD= Loss On Drying

2. Chromatographic purity By HpLC

Experimental /Chromatographic condition:

Equipment: Shimadzu LC-2010 HPLC system equipped with
a UV_VIS
detector or equivalent
Column: Inertsil C-8-3 ( 250 mm x 4.6 mm , 5 pm) Or equivalent
Wave length: 251rwr
Flow Rate: 0.8 ml/min
Column Temperature: 25"C
Injection volume: 10 pl
Run time: 30 minute

Reference standard or In house working standard

: Stage-III
 Preparation of Ammonium format buffer
solution :

lr*1*',3f,?,,ufi,f Ho"llif l,pHfi t"}ft mL volumetric flask and

1_00Q
$q5*-i'rffi"ffi Make.the volume up to the
mark with water and mix itE.o"tiir i ilt.ihril;i'bh pm lllter paper.
Preparation of Mobile phase:

Prepared a filter and degassed mixture of Buffer : Acetonitrile in the volume ratio of
950:50

Preparation of Diluent : Used DI water iis diluent

Preparation of standard solution:

weight accurately 25 mgof Stage-III standard transfer it into 50 mL

Add about l0 mL of *9:t
diluent inio the vilumetric flask, ,oni.ur. to
volumetric flask.
dissolve the content and
make up the volume up to the mark with diluent.
o.rig"r,. this solution as standard
solution. (500 pgl mL of Stage-Ilf

Preparation of sample solution:

weights accurately about 25 mgof test sample transfer

it into 50 mL volumetric flask. add
about Io' mL diluent into the volumetric flask, sonicate
to dissolve the content and make
up the volume up to the mark with diluent. Designate
this solution as standard solution.
(500 pgl mL of Stage-III)

Chromatographic procedure:

Sr.No. Sample Name No. of

01 Blank 0l
02 Standard Solution 01
03 Test Solution 01

The Approximate retention time for Stage-III is about

6.0 minutes,
Calculation:
Calculate the Impurities by area norTnalizationmethod
 3. Assay by titration ( On dry basis)

Apparatus:
Autotitrator, vorumetric flask, beaker, Measuring
cylinder, weighing balance

Reagent:
0.1 N perchloric acid
Glacial acetic acid
Acetic anhydride
potassium hydrogen phthalate

Preparation of 0.1 NHC1O4 VS:

Accurately transfer about 8.5 mL of Perchloric
acid (AR) to 1000 mL volumetric. Flask.
Add 500 mL glacial acetic acid and 2l mLacetic
anhydride, cool. Dilute to volume with
glacial acetic acid' Mix well allow the
solution to stand roi t aay for excess dcetic
anhydride to be combined and carry out
the determination of water, if water content
' exceeds 0'05yo, add more acetic anhydride.
If the solution contains no titratable water,
addsufficientwater toobtain acontentofwater
between 0.02% too.05o . Allowthe
solution to stand for one day and again titrate
the water content. The solution should
contain between 0'021 ando.o|%of water, indicatingfreedomfrom
acetic anhydride.

Standardization:

Dry the primary standard Potassium hydrogen phthalate

in oven at about 120"C for 2
hour' weigh accurately in dupricate about 0.35 g of previousry
dried potassium
hydrogen phthalate in titration cup. Transfer
50 mL orannya.ous glacial acetic acid
to each flask. Titrate against 0. r N perchloric
acid. Steepest jump in mi Iii volts
indicates the equivalence point of standard
ization. The Autotitrator itself
calculates titer value. Fof more details r.r.r
io op*;;;;i manual of Autotitrator

Normality N: W
vx tr

W: Weight of potassium hydrogen phthalate

v = volume of 0.1 N perchroric acidvS consumed
in the titration
F:204.23 mglml

Procedure:

weigh accurately about 200 mg sample and transfer it into

the titration vessel of an
autotitrator' Add about 60 mL of anhydrous acetic
acid this vessel and sonicate to dissolve
the contents' Titrate the resulting soluiion against
0.1 N perchloric acid. Determine the
end-point by Potentiometric titration using -a solvotrode
electrode. (Metrohm 6.0229.100,
Licl in ethanol) Similarly perform a blank titration.

Each ML 0.1 N perchloric acid is equivalent to r54.16 mg of stage-Ill

Calculation:

calculate the percentage of stage-rll by using

following formula:

7o Assay (On anhydrous basis): @

- Wx (100_ohLOD)

v : volume in mL of o.l N perchloric acid consumed

in the
titration (After blank correction)
N. : N-ormality of perchloric acid solution
w : Weight in mg of the test substance taken for the test determination.