6.Results and Discussion

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RESULTS AND DISCUSSION

Method Development

Trail 1:

Column : Symmetry C18 (4.6250mm)5

Column temperature : Ambient

Waelen!t" : 262 nm

#obile p"a$e ratio : #et"anol (100%)&'&

lo rate : 1ml'min

*n+e,tion olume : 10l

-un time : 8min

i!ure: ,"romato!ram or trail 1

Table: peak re!lt "or trail 1

S#No $eak Name R t Area %ei&htUS$

Tailin&

US$ $late

'o!nt

1 /ramaol1.50 1243 02

2.5 16

Obervation:

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*n t"i$ trial it $"o$ le$$ plate ,ount an more tailin! an improper $eparation o pea in t"e

,"romato!ram. So it reuire$ more trial$ to obtain !oo pea$.

Trail (:

Column : terra C18 (4.6250mm)5

Column temperature : 407C

Waelen!t" : 262 nm

#obile p"a$e ratio : #et"anol:Water (65:35 %)&'&

lo rate : 1ml'min


-un time : 10min

i!ure: ,"romato!ram or trail 2

Table: peak re!lt "or trail (

S#

No$eak name R t Area %ei&ht

US$

Tailin&

US$ plate

'o!nt

1 /ramaol 2.104 12668 6 2. 634

Obervation: *n t"i$ trial it $"o$ le$$ plate ,ount more plate ,ount an improper $eparation

o pea in t"e ,"romato!ram. So it reuire$ more trial$ to obtain !oo pea$.

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Trail ):

Column : *nert$il C18 (4.6250mm)5


Waelen!t" : 262nm

#obile p"a$e ratio : AC:Water: (20:80 %)&'&

lo rate : 1ml'min


-un time : 5min

i!ure; ,"romato!ram or trail 3

Table: * peak re!lt "or trail )

S# No $eak name R t Area %ei&htUS$

Tailin&

US$ plate

'o!nt

1 /ramaol 2.668 11542 635 2.14 335

Obervation: *n t"i$ trial it $"o$ le$$ plate ,ount more tailin! ee,t an improper

$eparation o pea in t"e ,"romato!ram. So it reuire$ more trial$ to obtain !oo pea$.

Trail +:

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Column :


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Column : ;/erra ->18 (4.6250mm) 5


Waelen!t" : 262nm

lo rate : 1ml'min


-un time : 5min

i!ure:


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i!ure:


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i&: Chromato&ram ho2in& blank -mobile phae preparation.

S3tem !itabilit3:

i&: Chromato&ram ho2in& in4e'tion *1

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i&: Chromato&ram ho2in& in4e'tion *(

i&: Chromato&ram ho2in& in4e'tion *)

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i&: Chromato&ram ho2in& in4e'tion *+

i&: Chromato&ram ho2in& in4e'tion *5

/able: -e$ult$ o $y$tem $uitability or /ramaol

S#No $eak Name RT

Area

-6/7e'.

%ei&ht

-6/. US$ $late Co!nt US$ Tailin&

1 /ramaol 2.13 505628 415 581 1.4

2 /ramaol 2.165 50584 454466 5810 1.4

3 /ramaol 2.12 506855 433328 5 1.60

4 /ramaol 2.15252066

430583 55 1.53

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5 /ramaol 2.128 501 4421 583 1.54

Mean 50648.6

Std# Dev# 6318.103

8 RSD 1.2

A''eptan'e 'riteria:

%-S= o ie ierent $ample $olution$ $"oul not more t"an 2

/"e %-S= obtaine i$ it"in t"e limit "en,e t"e met"o i$ $uitable.

S$ECIICIT9

/"e *C@ o,ument$ eine $pe,ii,ity a$ t"e ability to a$$e$$ uneuio,ally t"e analyte in t"e

pre$en,e o ,omponent$ t"at may be epe,te to be pre$ent $u," a$ impuritie$ e!raation

prou,t$ an matri ,omponent$.

Analyti,al met"o a$ te$te or $pe,ii,ity to mea$ure a,,urately uantitate /ramaol in

ru! prou,t.

Aa3 -Standard. :

i!. C"romato!ram $"oin! a$$ay o $tanar in+e,tion ;1

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Table: $eak re!lt "or aa3 tandard

S#No Name RT Area %ei&ht US$Tailin& U S$$late

Co!nt

In4e'tion

1 /ramaol 2.180 522 482601 1.44 585 1

2 /ramaol 2.168 55 4316 1.42 5814 2

3 /ramaol 2.16 564 48126 1.50 584 3

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Aa3 -Sample.:

i&: Chromato&ram ho2in& aa3 o" ample in4e'tion*1

i Chromato&ram ho2in& aa3 o" ample in4e'tion*(

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i&: Chromato&ram ho2in& aa3 o" ample in4e'tion*)

/able: >ea re$ult$ or A$$ay $ample

S#No Name RT Area %ei&ht US$Tailin& US$$late

Co!nt

In4e'tion

1 /ramaol 2.1 5661 46388 1.4 5820 1

2 /ramaol 2.184 53525 4600 1.50 5810 2

3 /ramaol 2.186 54005 463262 1.52 5841 3

%ASSAB

Sample area Wei!"t o $tanar =ilution o $ample >urity Wei!"t o tablet

DDDDDDDDDDD DDDDDDDDDDDDDDDD DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDD100

Stanar area =ilution o $tanar Wei!"t o $ample 100 Eabel ,laim

55431'566210'3030'0.0182.'1000.182'100100

100.8%

/"e % purity o /ramaol in p"arma,euti,al o$a!e orm a$ oun to be 100.8%%.

LINEARIT9

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i Chromato&ram ho2in& linearit3 level*1

i Chromato&ram ho2in& linearit3 level*(

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i Chromato&ram ho2in& linearit3 level*)

i Chromato&ram ho2in& linearit3 level*+

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i Chromato&ram ho2in& linearit3 level*5

C%ROMATORA$%IC DATA OR LINEARIT9 STUD9:

Con'entration

Level -8.

Con'entration

&;ml

Avera&e

$eak Area

33 20 168581

66 40 3540

100 60 52258

133 80 66532

166 100 8556

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LINEARIT9 $LOT:

/"e plot o Con,entration () er$u$ t"e Aera!e >ea Area (y) ata o /ramaol i$ a $trai!"t

line.

B m F ,

Slope (m) 8628

*nter,ept (,) 130

Correlation Coei,ient (r) 0.

/ALIDATION CRITERIA:/"e re$pon$e linearity i$ eriie i t"e Correlation Coei,ient i$

0. or !reater.

CONCLUSION: Correlation Coei,ient (r) i$ 0. an t"e inter,ept i$ 130. /"e$e alue$

meet t"e aliation ,riteria.

$re'iion:

/"e pre,i$ion o an analyti,al pro,eure epre$$e$ t"e ,lo$ene$$ o a!reement (e!ree o

$,atter) beteen a $erie$ o mea$urement$ obtaine rom multiple $amplin! o t"e $ame

"omo!eneou$ $ample uner t"e pre$,ribe ,onition$.

RE$EATA0ILIT9

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i Chromato&ram ho2in& pre'iion in4e'tion *)

i Chromato&ram ho2in& pre'iion in4e'tion *+

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i Chromato&ram ho2in& pre'iion in4e'tion *5

Table: Re!lt o" repeatabilit3 "or Tramadol:

S# No $eak nameRetention

timeArea-6/7e'.

%ei&ht

-6/.

US$ $late

Co!nt

US$

Tailin&

1 /ramaol 2.18 50041 446535806 1.55

2 /ramaol 2.14 50453 444485803 1.60

3 /ramaol 2.10 512545 440821585 1.60

4 /ramaol 2.1 505013 44658 1.56

5 /ramaol 2.12 506855 4333285 1.60

Mean 5018.2

Std#dev 3185.43

8RSD 0.62

A''eptan'e 'riteria:

%-S= or $ample $"oul be #/ 2

/"e %-S= or t"e $tanar $olution i$ belo 1 "i," i$ it"in t"e limit$ "en,e

met"o i$ pre,i$e.

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Intermediate pre'iion:

Anal3t 1:

i&: Chromato&ram ho2in& Anal3t 1 in4e'tion *1

i&: Chromato&ram ho2in& Anal3t 1 in4e'tion *(

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i&: Chromato&ram ho2in& Anal3t 1 in4e'tion *)

i&: Chromato&ram ho2in& Anal3t 1 in4e'tion *+

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i&: Chromato&ram ho2in& Anal3t 1 in4e'tion *5

i&: Chromato&ram ho2in& Anal3t 1 in4e'tion *3.345.'8628

2.86!'ml

!antitation limit

/"e uantitation limit o an iniiual analyti,al pro,eure i$ t"e loe$t amount o

analyte in a $ample "i," ,an be uantitatiely etermine.

LO>1=?@;S

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W"ere

G Stanar eiation o t"e re$pon$e

S Slope o t"e ,alibration ,ure

Re!lt: >1045.'8628

8.6!'ml

Rob!tne

/"e robu$tne$$ a$ perorme or t"e lo rate ariation$ rom 0. ml'min to 1.1ml'min an

mobile p"a$e ratio ariation rom more or!ani, p"a$e to le$$ or!ani, p"a$e ratio or

/ramaol. /"e met"o i$ robu$t only in le$$ lo ,onition an t"e met"o i$ robu$t een by

,"an!e in t"e #obile p"a$e H5%. /"e $tanar an $ample$ o /ramaol ere in+e,te by

,"an!in! t"e ,onition$ o ,"romato!rap"y. /"ere a$ no $i!nii,ant ,"an!e in t"e

parameter$ lie re$olution tailin! a,tor a$ymmetri, a,tor an plate ,ount.

/ariation in "lo2

i&!re: 'hromato&ram ho2in& le "lo2 o" =#Bml;min

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i&!re: 'hromato&ram ho2in& more "lo2 o" 1#1 ml;min

/ariation o" mobile phae or&ani' 'ompoition

i&!re: 'hromato&ram ho2in& le or&ani' 'ompoition

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i&!re: 'hromato&ram ho2in& more or&ani' 'ompoition

Table: Re!lt "or Rob!tne

$arameter !ed "or ample anal3i $eak Area Retention Time Theoreti'al

plateTailin& "a'tor

A,tual lo rate o 1.0 mE'min 56012 2.165 5846 1.41

Ee$$ lo rate o 0. mE'min 54838 2.8 5626 1.28

#ore lo rate o 1.1 mE'min 4855 1.2 532 1.33

#ore

Documents

6.Results and Discussion