Sarthak Biotech Pvt. LTD.: Assay Method Validation Name of The Product: Sarcip-500 Tablet

Sarthak Biotech Pvt. Ltd.
Total Page: 01-06

Assay Method Validation QC/VLD/01
Name of the Product: SARCIP-500 TABLET
Prepared By Checked By/ Approved By
Date of Issue/Effective Date : 18/11/2007 Review Date : 17/11/2009 Revision No. : 00
OBJECTIVE : The establish documented evidence which will provide a high degree of
assurance that the analytical method for assay of Ciprofloxacin in Sarcip-
500 Tablet will give results with acceptable Accuracy, Linearity,
Repeatibility, Relative Standard Deviation & Ruggedness when applied to
the Sarcip-500 Tablet.
SCOPE : This method applies to the procedure which is used for calculation and assay
of Ciprofloxacin.
RESPONSIBILITY: 1. Quality Control Manager.
2. Asstt. Quality Control Chemist.
VALIDATION PARAMETERS:
1. Linearity
2. Repeatibility.
3. Relative Standard Deviation.
4. Ruggedness.
Sarthak Biotech Pvt. Ltd. Total Page: 02-06
Date of Issue/Effective Date : 18/11/2007Review Date : 17/11/2009 Revision No. : 00
METHOD :
1. Weight accurately equivalent to about 100mg of Ciprofloxacin working Reference

standard and its equivalent to test in a 100ml volumetric flask . Dissolve with 0.1N-
Hydrochloric Acid and make up to same, mix and filter if required.
2. With above dilution prepare standard and sample solution of 100mcg/ml with 0.1N
Hydrochloric acid separately, mix and make homogeneous solution.
3. Pipette 3ml,5ml,7ml,9ml and 11ml of standard and sample solution separately in a

calibrated 50ml.volumetric flask.
4. To each flask add 1ml of 5%w\v solution of Anhydrous Ferric chloride in water. Make
up with distilled water to produced 50ml, mix to make homogeneous solution.
5. Measure the absorbance at maximum at about 438nm,against water as blank and

tabulate the observed data in the observation table.
6. Plot linearity curve from the different concentration corresponding area of

Ciprofloxacin peak. Taking concentration on X-axis and corresponding peak on Y-
axis. Plot graph should be connected at least three points.
7. Plot repeatability curve from by taking Ciprofloxacin solution serial no. on X-axis and
observed peak area on Y-axis.
8. Draw a line which should cover almost all the points. The resulted graph should be
almost parallel to the X-axis. . .
9. Calculated the result by comparison with peak area obtained in standard and sample
solution respectively.
LINEARITY STUDY
The linearity of an analytical method is its ability to elicit test results data directly proportional
to the concentration of the analyte in samples within given range.
Linearity of assay method of Ciprofloxacin in Sarcip-500 Tables shall be studied by measuring

the absorbance of the standard Ciprofloxacin solution of 20 – 100mcg/ml concentration &
range 438nm. Linearity curve shall be plotted for absorbance against concentration.
OBSERVATION TABLE FOR LINEARITY
Sr.No. Concentration of Ciprofloxacin in mcg\ml Peak area
1. 20mcg\ml 405
2. 40mcg\ml 450
3. 60mcg\ml 535
4. 80mcg\ml 570
5. 100mcg\ml 605
REPEATIBILITY STUDY
Repeatibility of assay method of Ciprofloxacin in Sarcip-500 Tables shall be studied by

measuring the absorbance of the standard Ciprofloxacin solution of 100mcg/ml concentration
& range 438nm. Repeatibility curve shall be plotted for absorbance against concentration.
OBSERVATION TABLE FOR REPEATIBILITY
Sr. No. Concentration in mcg\ml Peak area
1 100mcg\ml 604
2. 100mcg\ml 608
3. 100mcg\ml 603
4. 100mcg\ml 605
5. 100mcg\ml 604

RELATIVE STANDARD DEVIATION
Test concentration of Ciprofloxacin used = 100mcg\ml.
Number of test solution used = 5
604+608+603+605+604
Average area = ----------------------------------- = 604.8
5
(604-604.8)2 +(608-604.8)2 +(603-604.8)2 +(605-604.8)2 +(604-604.8)2

Dev. S =  -------------------------------------------------------------------------------------- 1/2
5-1
0.64+10.24+3.24+0.04+0.64
= --------------------------------------1/2
4
14.8
=  -------1/2
4
=  3.7 1/2
= 1.9235
1.9235 x 100
RSD = ------------------ = 0.318%
604.8
Limit : ( Not more than 2.0% )

RUGGEDNESS STUDY
The ruggedness of an analytical method is the degree of reproducibility of the test

results obtained by the analysis of the samples from same lot under a variety of normal test
conditions (i.e. different laboratories, analysts, days & Instruments).
The ruggedness of assay method of Ciprofloxacin in Sarcip-500 tablet shall be studied
by analyzing the samples from same lot by two different analysts from same laboratory or at
two different laboratories by two different analysts on two different days.
RUGGEDNESS STUDY DATA
Ruggedness data for assay method by two different analysts on different days and
different instrument.
Sr. Analyst- I (Rajnish Kohli) Analyst- II (Satpal Saini)

No. Date of analysis : Date of analysis :
Instrument & Model : Instrument & Model :
UV Spectrophotometer, Systronics 117 UV Spectrophotometer, Systronics 117
Location : Instrument Lab. Location : Instrument Lab.
Wt. of Wt. of Measured Measured % of Wt. of Wt. of Measured Measured % of
Ref. Sample Absor- Absor- Cipro- Ref. Sample Absor- Absor- Cipro-
Standard gm. bance at bance at floxacin Standard gm bance at bance at floxacin
gm. of Ref. 438nm of gm. of Ref. 438nm
Std. sample Std. of sample
01 0.1168 0.1348 602 608 100.11 0.1162 0.1349 606 601 99.43
02 0.1156 0.1352 607 612 99.61 0.1165 0.1356 609 616 101.15
03 0.1158 0.1342 605 615 101.35 0.1164 0.1352 612 608 99.55
Average wt of tablet 670.2 mg Averagewt.of tablet 675.1 mg
Mean 100.35% Mean 100.04%
Standard Deviation 10% Standard Deviation 10%
Combining mean of I & II of both the analysts :
Mean : 100.2 %
Standard Deviation : ±10 %
Name of the Product: SARFLOK-200 TABLET
PURPOSE :-The establish documented evidence which will provide a high degree of
assurance that the analytical method for assay of Ofloxacin in Sarflok-200
Tablet will give results with acceptable Accuracy, Linearity, Repeatibility,
Relative Standard Deviation & Ruggedness when applied to the Sarflok-200
Tablet.
SCOPE:- This method applies to the procedure which is used for calculation and assay
of Ofloxacin.
VALIDATION PARAMETERS :-
1. Linearity.
2. Repeatibility.
4. Ruggedness.
METHOD
1. Weigh accurately equivalent to about 50mg of Ofloxacin working Reference

standard and its equivalent to test in a 100ml volumetric flask . Dissolve with 0.1N-
Hydrochloric Acid and make up to same, mix and filter if required.

4. To each flask add 1ml of 5%w\v solution of Ferric Chloride. Make up the volume with
distilled water to produce 50ml, mix to make homogeneous solution.
5. Measure the absorbance at maximum at about 437nm,against water as blank and

calculate the observed data in the observation table.
6. Plot linearity curve from the different concentration corresponding area of Ofloxacin
peak. Taking concentration on X-axis and corresponding peak on Y-axis .Plot graph
should be connected at least three points.
7. Plot repeatability curve from by taking Ofloxacin solution serial no. on X-axis and
LINEARITY STUDY
Linearity of assay method of Ofloxacin in Sarflox-200 Tables shall be studied by measuring the
absorbance of the standard Ofloxacin solution of 30 – 70mcg/ml concentration & range
437nm. Linearity curve shall be plotted for absorbance against concentration.
Sr.No. Concentration of Ofloxacin in mcg\ml Peak area
1. 30mcg\ml 368
2. 40mcg\ml 437
3. 50mcg\ml 512
4. 60mcg\ml 591
5. 70mcg\ml 664
REPEATIBILITY STUDY
Repeatibility of assay method of Ofloxacin in Sarflox-200 Tables shall be studied by

measuring the absorbance of the standard Ofloxacin solution of 50 mcg/ml concentration &
range 437nm.Repeatibility curve shall be plotted for absorbance against concentration.
S.No. Concentration in mcg\ml Peak area
1 50mcg\ml 515
2. 50mcg\ml 512
3. 50mcg\ml 515
4. 50mcg\ml 509
5. 50mcg\ml 519

Test concentration of Ofloxacin used = 50mcg\ml.
515 + 512 + 515 + 509 + 519

Average area = ------------------------------------------ = 513.4
5
(515-513.4)2 +(512-513.4)2 +(515-513.4)2 +(509-513.4)2 +(519-513.4)2 1/2
Dev. S =  ---------------------------------------------------------------------------------------
5-1
2.56+1.96+1.96+19.36+31.36
= ---------------------------------------1/2
4
57.2
=  ------- 1/2
4
=  14.3 1/2
= 3.781
3.781 x 100
RSD = ------------------ = 0.736%
513.4

RUGGEDNESS STUDY

The ruggedness of assay method of Ofloxacin in Sarflok-200 tablet shall be studied by
analyzing the samples from same lot by two different analysts from same laboratory or at two
different laboratories by two different analysts on two different days.
Sr. Analyst- I (Satpal Saini) Analyst- II (Rajnish Kohli)

Ref. Sample Absorbanc Absorbance Ofloxacin Ref. Sample Absorbanc Absor- Ofloxacin
Standard gm. e at at 437nm Standard gm e at bance at
gm. of Ref. of sample gm. of Ref. 437nm
Std. Std. of sample
01 0.0498 0.1021 509 512 100.67 0.0499 0.1006 512 505 98.97
02 0.0501 0.1019 511 515 101.7 0.0512 0.1018 504 510 102.95
03 0.0512 0.1029 519 512 100.7 0.0489 0.1012 508 514 98.90
Mean 101.02% Mean 100.27%
Mean : 100.65 %

Name of the Product: SARNIM-P TABLET
PURPOSE : The establish documented evidence which will provide a high degree of
assurance that the analytical method for assay of Nimesulide & Paracetamol in
Sarnim-P Tablet will give results with acceptable Accuracy, Linearity,
Repeatibility, Relative Standard Deviation & Ruggedness when applied to the
Sarnim-P Tablet.
SCOPE : This method applies to the procedure which is used for calculation and assay
of Paracetamol & Nimesulide.
RESPONSIBILITY : 1. Quality Control Manager.
1. Linearity.
2. Repeatibility.
4. Ruggedness.

PROCEDURE :
(For Paracetamol)
1. Weight accurately equivalent to about 60mg of Paracetamol working Reference

standard and its equivalent to sample in a 100ml volumetric flask . Dissolve with 20ml
of Hydrochloric Acid 1M and digest for 30 minutes, then make up the volume 100ml
with distilled water.

calibrated 50ml. measuring flask.
3. To each flask add 2ml of 10%w\v solution of Sodium Nitrate & allow to stand for 5
minutes, then add 2ml of 10% w/v solution of Ammonium Sulphamate. Again allow to
stand for 5 to 6 minutes now further add 2 ml. of 10%w/v solution of Sodium
Hydroxide and keep it for 5 minutes. Now adjust the volume to 50ml with distilled
water.
4. Measure the absorbance at maximum at about 450nm, against water as blank and
5. Plot linearity curve from the different concentration corresponding area of Paracetamol
6. Plot repeatability curve from by taking Paracetamol solution serial no. on X-axis and

LINEARITY STUDY
Linearity of assay method of Paracetamol in Sarnim-P Tables shall be studied by measuring the
absorbance of the standard Sarnim-P solution of 24 – 72mcg/ml concentration & range 450nm.
Linearity curve shall be plotted for absorbance against concentration.
Sr.No. Concentration of Paracetamol in mcg\ml Peak area
1. 24mcg\ml 525
2. 36mcg\ml 595
3. 48mcg\ml 645
4. 60mcg\ml 695
5. 72mcg\ml 742

REPEATIBILITY STUDY
Repeatibility of assay method of Paracetamol in Sarnim-P Tables shall be studied by measuring

the absorbance of the standard Sarnim-P solution of 24 mcg/ml concentration & range 450nm.
Repeatibility curve shall be plotted for absorbance against concentration.
OBSERVATION TABLE FOR REPEATBILITY
Sr.No. Concentration in mcg\ml Peak area
1 24mcg\ml 525
2. 24mcg\ml 528
3. 24mcg\ml 527
4. 24mcg\ml 526
5. 24mcg\ml 525

Test concentration of Paracetamol used = 24mcg\ml.
525 + 528 + 527 + 526 + 525

Average area = ------------------------------------------ = 526.2
5
(525-526.2)2 +(528-526.2)2 +(527-526.2)2 +(526-526.2)2 +(525-526-2)2 1/2

Dev. S =  ---------------------------------------------------------------------------------------
5-1
1.44+3.24 +0.64+0.04+1.44
= ---------------------------------------1/2
4
6.8
=  -------1/2
4
=  1.7 1/2
= 1.303
1.303 x 100
RSD = ------------------ = 0.2477%
526.2

RUGGEDNESS STUDY

The ruggedness of assay method of Paracetamol in Sarnim-P tablet shall be studied by

Ref. Sample Absor- Absor- Paraceta Ref. Sample Absor- Absor- Paraceta-
Standard gm. bance at bance at mol-mol Standard gm bance at bance at mol
01 0.0598 0.0784 521 528 100.83 0.0602 0.0782 512 518 101.99
02 0.0601 0.0792 532 528 98.23 0.0592 0.0779 519 515 98.75
03 0.0592 0.0784 518 525 99.19 0.0612 0.0791 522 525 101.9
Mean 99.41% Mean 100.88%
Mean : 100.15 %

PROCEDURE :
(For Nimesulide)
1. Weight accurately equivalent to about 50mg of Nimesulide working Reference

standard and its equivalent to sample in a 100ml volumetric flask . Dissolve with 30 to
40ml of 0.1M solution of Sodium Hydroxide. Digest both the solution for 10 minutes
then make up the volume to 100ml with 0.1M solution of Sodium Hydroxide.

calibrated 50ml.measuring flask.
3. To each flask add 20ml 0.1M Sodium Hydroxide solution shake well and further adjust
the volume up to 50ml with 0.1M solution of Sodium Hydroxide.
4. Measure the absorbance of both the solution at 405nm, against 0.1M Sodium
Hydroxide solution as blank and tabulate the observed data in the observation table.
5. Plot linearity curve from the different concentration corresponding area of Nimesulide
6. Plot repeatability curve from by taking Nimesulide solution serial no. on X-axis and

LINEARITY STUDY
Linearity of assay method of Nimesulide in Sarnim-P Tablets shall be studied by measuring the
absorbance of the standard Nimesulide solution of 20 – 60mcg/ml concentration & range
Sr.No. Concentration of Nimesulide in mcg\ml Peak area
1. 20mcg\ml 552
2. 30mcg\ml 595
3. 40mcg\ml 635
4. 50mcg\ml 702
5. 60mcg\ml 757

REPEATBILITY STUDY
Repeatibility assay method of Nimesulide in Sarnim-P Tablets shall be studied by measuring

the absorbance of the standard Nimesulide solution of 20 mcg/ml concentration & range
405nm. Repeatibility curve shall be plotted for absorbance against concentration.
1 20mcg\ml 559
2. 20mcg\ml 561
3. 20mcg\ml 561
4. 20mcg\ml 560
5. 20mcg\ml 563

Test concentration of Nimesulide used = 20mcg\ml.
559 + 561 + 561 + 560 + 563

Average area = ------------------------------------------ = 560.8
5
(559-560.8)2 +(561-560.8)2 +(561-560.8)2 +(560-560.8)2 +(563-560.8)2 1/2

Dev. S =  ---------------------------------------------------------------------------------------
5-1
3.24+0.04 +0.04+0.64+4.84
= ---------------------------------------1/2
4
8.8
=  -------1/2
4
=  2.2 1/2
= 1.4142
1.4142 x 100
RSD = ------------------ = 0.2521%
560.8

RUGGEDNESS STUDY

The ruggedness of assay method of Nimesulide in Sarnim-P tablet shall be studied by

Ref. Sample Absor- Absor- Nimesuli Ref. Sample Absor- Absor- Nimesuli
Standard gm. bance at bance at de Standard gm bance at bance at de
01 0.0499 0.3258 560 568 101.31 0.0498 0.3261 565 560 99.11
02 0.0489 0.3251 542 545 98.64 0.0496 0.3269 559 562 99.88
03 0.0501 0.3251 560 555 99.61 0.0502 0.3251 553 556 101.65
Mean 99.85% Mean 100.21%
Mean : 100.03 %

Name of the Product: SARFIX-100 DT TABLET
PURPOSE :- The establish documented evidence which will provide a high degree of
assurance that the analytical method for assay of Cefixime Trihydrate in Sarfix-
100 DT Tablet will give results with acceptable Accuracy, Linearity,
Repeatability, Relative Standard Deviation & Ruggedness when applied to the
Sarfix-100 DT Tablet.
SCOPE:- This method applies to the procedure which is used for calculation and assay
of Cefixime Trihydrate.
VALIDATION PARAMETERS :-
1. Linearity.
2. Repeatibility.
4. Ruggedness.

METHOD
1. Weigh accurately equivalent to about 100mg of Cefixime working Reference standard

and its equivalent to test in a 100ml volumetric flask . Dissolve with Methanol and
make up to volume up to 100ml.

3. To each flask add Methanol and make up the volume up to 100ml.
4. Measure the absorbance at maximum at about 268nm,against Methanol as blank and

calculate the observed data in the observation table.
5. Plot linearity curve from the different concentration corresponding area of Cefixime
6. Plot repeatability curve from by taking Cefixime solution serial no. on X-axis and

LINEARITY STUDY
Linearity of assay method of Cefixime Trihydrate in Sarfix-100 DT Tablets shall be studied by

measuring the absorbance of the standard Cefixime Trihydrate solution of 20 – 50mcg/ml
concentration & range 268nm. Linearity curve shall be plotted for absorbance against
concentration.
Sr.No. Concentration of Cefixime in mcg\ml Peak area
1. 10mcg\ml 495
2. 20mcg\ml 515
3. 30mcg\ml 540
4. 40mcg\ml 570
5. 50mcg\ml 595

REPEATIBILITY STUDY
Repeatibility of assay method of Cefixime Trihydrate in Sarfix-100 DT Tablets shall be
studied by measuring the absorbance of the standard Cefixime Trihydrate solution of 10mcg/ml
concentration & range 268nm. Repeatibility curve shall be plotted for absorbance against
concentration
1 10mcg\ml 495
2. 10mcg\ml 492
3. 10mcg\ml 497
4. 10mcg\ml 495
5. 10mcg\ml 496

Test concentration of Cefixime used = 10mcg\ml.
495 + 492 + 497 + 495 + 496

Average area = ------------------------------------------ = 495
5
(495-495)2 +(495-492)2 +(495-497)2 +(495-495)2 +(495-496)2 1/2
Dev. S =  --------------------------------------------------------------------------
5-1
0+9+4+0+1
= ---------------------------------------1/2
4
14
=  ------- 1/2
4
=  3.5 1/2
= 1.8708
1.8708 x 100
RSD = ------------------ = 0.377%
495

RUGGEDNESS STUDY
The ruggedness of assay method of Cefixime Trihydrate in Sarfix-100 DT Tablet shall
be studied by analyzing the samples from same lot by two different analysts from same
laboratory or at two different laboratories by two different analysts on two different days.

Ref. Sample Absor- Absor- Cefixime Ref. Sample Absor- Absor- Cefixime
Standard gm. bance at bance at Standard gm bance at bance at
01 0.1128 0.4019 498 502 101.75 0.1118 0.4028 502 508 101.21
02 0.1119 0.4021 492 499 101.50 0.1129 0.4031 499 505 102.14
03 0.1131 0.4012 498 488 99.34 0.1125 0.4022 492 498 102.02
Mean 100.86% Mean 101.79%
Mean : 101.32 %

Name of the Product: SARMOX-500 CAPSULE
PURPOSE: The establish documented evidence which will provide a high degree of
assurance that the analytical method for assay of Amoxycillin in Sarmox-500
Capsule will give results with acceptable Accuracy, Linearity, Repeatibility,
Relative Standard Deviation & Ruggedness when applied to the Sarmox-500
Capsule.
SCOPE : This method is applies to the procedure which is used for calculation and assay
of Amoxycillin.
1. Linearity.
2. Repeatability.
4. Ruggedness.

METHOD :
1. Weight accurately 100mg Reference standard and equivalent to 100mg. Amoxycillin as

test sample. Bowl it with 20 to 30ml of distilled water and make up to produce 100ml.
2. Pipette 2ml, 3ml, 4ml, 5ml and 6ml of standard and sample solution separately in a
3. In a 50ml volumetric flask pipette 2ml Reference and test solution separately. To
each flask add 2ml of Phosphate buffer pH 9.2 and add 1ml Vitamin B6 dye..
4. Measure the absorbance without delay at maximum at about 605nm,against respective

blank and tabulate the observed data in the observation table.
5. Plot linearity curve from the different concentration corresponding area of Amoxycillin
peak. Taking concentration on X-axis and corresponding peak on Y-axis. Plot graph
6. Plot repeatability curve from by taking Amoxycillin solution serial no. on X-axis and

LINEARITY STUDY
Linearity of assay method of Amoxycillin in Sarmox-500 Capsule shall be studied by
measuring the absorbance of the standard Amoxycillin solution of 40 – 120mcg/ml
concentration.
Sr.No. Concentration of Amoxycillin in mcg/ml Peak area of
1. 40mcg\ml 652
2. 60mcg\ml 695
3. 80mcg\ml 728
4. 100mcg\ml 765
5. 120mcg\ml 805

REPEATIBILITY STUDY
Repeatibility of assay method of Amoxycillin in Sarmox-500 Capsule shall be studied by
measuring the absorbance of the standard Amoxycillin solution of 40mcg/ml concentration &
range 605nm. Repeatibility curve shall be plotted for absorbance against concentration.
OBSERVATION TABLE FOR REPEATBILITY

1 40mcg\ml 651
2. 40mcg\ml 653
3. 40mcg\ml 652
4. 40mcg\ml 657
5. 40mcg\ml 651


Test concentration of Amoxycillin used = 40mcg\ml.
651+653+652+657+651
Average area = ----------------------------------- = 652.8
5
(651-652.8)2 +(653-652.8)2 +(652-652.8)2 +(657-652.8)2 +(651-652.8)2

Dev. S =  ------------------------------------------------------------------------------------- 1/2
5-1
3.24+0.04+0.64+17.64+3.24
= -------------------------------------1/2
4
24.8
=  -------1/2
4
=  6.2  1/2
= 2.489
2.489 x 100
RSD = ------------------ = 0.381%
652.8

RUGGEDNESS STUDY
The ruggedness of assay method of Amoxycillin in Sarmox-500 Capsule shall be
studied by analyzing the samples from same lot by two different analysts from same laboratory
or at two different laboratories by two different analysts on two different days.

Ref. Sample Absor- Absor- Amoxyci Ref. Sample Absor- Absor- Amoxycil
Standard gm. bance at bance at -llin Standard gm bance at bance at -lin
01 0.1162 0.1148 655 652 100.79 0.1154 0.1168 659 655 98.57
02 0.1159 0.1162 654 658 100.38 0.1162 0.1169 652 662 101.30
03 0.1165 0.1155 653 657 101.51 0.1152 0.1162 656 650 98.60
Average fill weight 580.2 mg Average fill weight 582.2 mg
Mean 100.89% Mean 99.49%
Mean : 100.19 %

Name of the Product: ACIGONE CAPSULE
Date of Issue/Effective Date :27/09/2007 Review Date : 26/09/2009 Revision No. : 00
assurance that the analytical method for assay of Omeprazole in Acigone
Capsule will give results with acceptable Accuracy, Linearity, Repeatability,
Relative Standard Deviation & Ruggedness when applied to the Acigone
Capsule.
of Omeprazole.
1. Linearity.
2. Repeatability.
4. Ruggedness.

METHOD :
1. Weight accurately 20mg Reference standard and equivalent to 20mg. Omeprazole as

test sample. Dissolve it with 20 to 30ml of 0.1M Sodium Hydroxide solution shake
vigorously 4-5 minutes and make up the volume up to 50ml with 0.1M NaOH solution.
3. In a 50ml volumetric flask pipette 1ml Reference and test solution separately and make
up the volume up to 50ml. with 0.1M Sodium Hydroxide solution.

5. Plot linearity curve from the different concentration corresponding area of Omeprazole
6. Plot repeatability curve from by taking Omeprazole solution serial no. on X-axis and

LINEARITY STUDY
Linearity of assay method of Omeprazole in Acigone Capsule shall be studied by measuring
the absorbance of the standard Omeprazole solution of 8 – 40mcg/ml concentration & range
Sr.No. Concentration of Amoxycillin in mcg\ml Peak area of
1. 8mcg\ml 397
2. 16mcg\ml 421
3. 24mcg\ml 495
4. 32mcg\ml 565
5. 40mcg\ml 605

REPEATIBILITY STUDY
Repeatibility of assay method of Omeprazole in Acigone Capsule shall be studied by
measuring the absorbance of the standard Omeprazole solution of 8 mcg/ml concentration &

1 8mcg\ml 398
2. 8mcg\ml 401
3. 8mcg\ml 397
4. 8mcg\ml 399
5. 8mcg\ml 398


398+401+397+399+398
Average area = ----------------------------------- = 398.6
5
(398-398.6)2 +(401-398.6)2 +(397-398.6)2 +(399-398.6)2 +(398-398.6)2

Dev. S =  --------------------------------------------------------------------------------------- 1/2
5-1
0.36+5.76 +2.56+0.16+0.36
= ---------------------------------------1/2
4
9.2
=  -------1/2
4
=  2.3  1/2
= 1.5165
1.5165 x 100
RSD = ------------------ = 0.3804%
398.6

RUGGEDNESS STUDY
The ruggedness of assay method of Omeprazole in Acigone Capsule shall be studied

Ref. Sample Absor- Absor- Omepraz Ref. Sample Absor- Absor- Omepra-
Standard gm. bance at bance at -ole Standard gm bance at bance at zole
01 0.2657 0.2940 395 399 100.40 0.2672 0.2926 403 399 99.48
02 0.2673 0.2927 401 396 99.20 0.2657 0.2961 398 401 99.47
03 0.2661 0.2939 393 396 100.38 0.2673 0.2926 396 393 99.75
Average fill weight 293.4 mg Average fill weight 293.4 mg
Mean 99.99% Mean 99.57%
Mean : 99.78
Standard Deviation : ±10%

Name of the Product: SARMOX DRY SYRUP
assurance that the analytical method for assay of Amoxycillin in Sarmox Dry
Syrup will give results with acceptable Accuracy, Linearity, Repeatability,
Relative Standard Deviation & Ruggedness when applied to the Sarmox Dry
Syrup.
of Amoxycillin.
1. Linearity.
2. Repeatibility.
4. Ruggedness.

METHOD :
1. Weight accurately 100mg Reference standard and equivalent to 100mg. Amoxycillin as

test sample. Bowl it with 20 to 30ml of distilled water and make up to produce 100ml.
3. In a 50ml volumetric flask pipette 2ml Reference and test solution separately. To
each flask add 2ml of Phosphate buffer pH 9.2 and add 1ml Vitamin B6 dye. And make
up the volume up to 50ml with water.

5. Plot linearity curve from the different concentration corresponding area of Amoxycillin
6. Plot repeatability curve from by taking Amoxycillin solution serial no. on X-axis and

LINEARITY STUDY
Linearity of assay method of Amoxycillin in Sarmox Dry Syrup shall be studied by measuring
the absorbance of the standard Amoxycillin in solution of 40 – 120mcg/ml concentration &
Sr.No. Concentration of Amoxycillin in mcg\ml Peak area of
1. 40mcg\ml 651
2. 60mcg\ml 695
3. 80mcg\ml 726
4. 100mcg\ml 775
5. 120mcg\ml 804

REPEATIBILITY STUDY
Repeatibility of assay method of Amoxycillin in Sarmox Dry Syrup shall be studied by
measuring the absorbance of the standard Amoxycillin in solution of 40 mcg/ml concentration
& range 605nm. Repeatibility curve shall be plotted for absorbance against concentration.

1 40mcg\ml 651
2. 40mcg\ml 652
3. 40mcg\ml 657
4. 40mcg\ml 660
5. 40mcg\ml 655


651+652+657+660+655
Average area = ----------------------------------- = 655
5
(651-655)2 +(652-655)2 +(657-655)2 +(660-655)2 +(655-655)2

Dev. S =  ------------------------------------------------------------------------------ 1/2
5-1
16+ 9+ 4 + 25 + 0
= ---------------------------------------1/2
4
54
=  -------1/2
4
=  13.5  1/2
= 3.6742
3.6742 x 100
RSD = ------------------ = 0.560%
655

RUGGEDNESS STUDY
The ruggedness of assay method of Amoxycillin in Sarmox Dry Syrup shall be studied

Wt. of Wt. of Measure Measured % of Wt. of Wt. of Measured Measured % of
Ref. Sample d Absor- Amoxy- Ref. Sample Absor- Absor- Amoxy-
Standard ml Absor- bance at cillin Standard ml bance at bance at cillin
gm. bance 605nm of gm. of Ref. 605nm
at sample Std. of sample
of Ref.
Std.
01 0.1161 4ml 653 656 100.54 0.1159 4ml 657 652 99.15
02 0.1168 4ml 658 665 101.76 0.1162 4ml 659 663 100.18
03 0.1155 4ml 652 656 100.82 0.1155 4ml 657 654 99.11
Label Claim 125mg Amoxycillin/ 5ml Label Claim 125mg Amoxycillin/ 5ml
Mean 100.82% Mean 100.01%

Mean : 100.41 %

Name of the Product: CEFSAR-125 DRY SYRUP
assurance that the analytical method for assay of Cephalexin in Cefasar-125 Dry
Syrup will give results with acceptable Accuracy, Linearity, Repeatibility,
Relative Standard Deviation & Ruggedness when applied to the Cefasar-125
Dry Syrup.
of Cephalexin.
1. Linearity;
2. Repeatibility.
4. Ruggedness.

METHOD :
1. Weight accurately equivalent 250mg of Cephalexin and dissolve in 20ml of Neutral

Methanol and titrate with 0.1M Sodium Hydroxide using Bromomethyl blue as
indicator, perform a blank for necessary correction. Each ml. of 0.1M Sodium
Hydroxide is equivalent to 0.03474gm. of Cephalexin.
2. Weight 250mg, 300mg, 350mg., 400mg. & 450mg. Cephalexin in 20ml. of Neutral
Methanol & titrate with 0.1M NaOH using Bromomethyl blue as indicator. Each ml. of
0.1m Sodium Hydroxide is equivalent to 0.03474gm. of Cephalexin.
3. Note the volume consumed & tabulate the observed data in observation table.
4. Plot linearity curve from the different concentration corresponding area of Cephalexin
5. Plot repeatability curve from by taking Cephalexin solution serial no. on X-axis and
7. Calculated the result by calculation volume consumed by the Cephalexin.

LINEARITY STUDY
Linearity of assay method of Cephalexin in Cefsar-125 Dry Syrup shall be studied by 250mg-
to 450 mg Cephalexin.
( Molarity of 0.1M NaOH is 0.102305)
Sr.No. Weight of Cephalexin in mg Volume consumed
1. 250mg. 7ml
2. 300mg. 8.6ml
3. 350mg. 9.7ml
4. 400mg. 11.2ml
5. 450mg. 12.7ml

REPEATIBILITY STUDY
Repeatibility of assay method of Cephalexin in Cefsar-125 Dry Syrup shall be studied by 250
mg Cephalexin.
( Molarity of 0.1M NaOH is 0.102305)
Sr.No. Weight of Cephalexin Volume consumed
1 250mg. 7ml
2. 250mg. 6.9ml
3. 250mg. 7.1ml
4. 250mg. 7.2ml
5. 250mg. 6.9ml

Test concentration of Cephalexin used = 250mg.
7+6.9+7.1+7.2+6.9
Average volume = ----------------------------------- = 7.02
5
(7- 7.02)2 +(6.9-7.02)2 +(7.1-7.02)2 +(7.2-7.02)2 +(6.9-7.02)2

Dev. S =  --------------------------------------------------------------------------------------- 1/2
5-1
0.0004+0.0144+0.0064+0.0324+0.0144
= ------------------------------------------------------1/2
4
0.068
=  ------- 1/2
4
=  0.017  1/2
= 0.130
0.130 x 100
RSD = ------------------ = 1.857%
7.02

RUGGEDNESS STUDY

The ruggedness of assay method of Cephalexin inCefsar-125 Dry Syrup shall be

Burette 50ml (Borosil) Burette 50ml (Borosil)
Location : Chemical Lab. Location : Chemical Lab.
Wt. of Molarity of Final Assay Wt. of Molarity of Final Volume Assay
Sample 0.1M Volume Sample 0.1M used
NaOH used NaOH
01 10ml 0.102305 7.0ml 99.51 10ml 0.102305 7.1ml 100.94
02 10ml 0.102305 6.9ml 98.09 10ml 0.102305 7.0ml 99.51
03 10ml 0.102305 7.0ml 99.51 10ml 0.102305 7.0ml 99.52
Label Claim 125mg Cephalexin/5ml Label Claim 125mg Cephalexin/5ml
Mean 99.04% Mean 99.99%
Mean : 99.52%

Name of the Product: CHLORTEX MOUTH WASH
assurance that the analytical method for assay of Chlorhexidine Gluconate in
Chlortex Mouth Wash will give results with acceptable Accuracy, Linearity,
Repeatability, Relative Standard Deviation & Ruggedness when applied to the
Cefasar-125 Dry Syrup.
of Chlorhexidine Gluconate.
1. Linearity.
2. Repeatability.
4. Ruggedness.

METHOD :
1. Weigh accurately equivalent of 200mg. of Chlorhexidine Gluconate Reference
standard and dissolve in 100ml of distill water.
2. Further pipette 5ml, 10ml, 15ml, 20ml & 25ml of standard and dilute to 100ml. with
water.
3. Weigh accurately equivalent to 5mg., 10mg., 15mg., 20mg. & 25mg. of sample,
basified with 10M NaOH and extracted with Chloroform (2 x 20ml). Combine
Chloroform layer and dilute to 50ml with water. Further prepare dilution of 50mcg / ml.
with water.
4. To 1 ml. of each above standard and sample dilutions add 3ml of dye solution and 5ml.
of Chloroform. Shake well for 3 to 4 minutes.
5. Measure the absorbance without delay at maximum at about 410nm, against respective

Chlorhexidine Gluconate peak. Taking concentration on X-axis and corresponding
peak on Y-axis. Plot graph should be connected at least three points.
7. Plot repeatability curve from by taking Chlorhexidine Gluconate solution serial no. on
X-axis and observed peak area on Y-axis.

LINEARITY STUDY
Linearity of assay method of Chlorhexidine Gluconate in Chlortex Mouthwash shall be studied

by measuring the absorbance of the standard Chlorhexidine Gluconate in solution of 100 –
500mcg/ml concentration & range 410nm. Linearity curve shall be plotted for absorbance
against concentration.
Sr.No. Concentration of Chlorhexidine Gluconate in mcg\ml Peak area of
1. 100mcg\ml 546
2. 200mcg\ml 642
3. 300mcg\ml 726
4. 400mcg\ml 791
5. 500mcg\ml 805

REPEATIBILITY STUDY
Repeatibility of assay method of Chlorhexidine Gluconate in Chlortex Mouthwash shall be
studied by measuring the absorbance of the standard Chlorhexidine Gluconate in solution of
100 mcg/ml concentration & range 410nm. Repeatibility curve shall be plotted for absorbance
against concentration
1 100mcg\ml 544
2. 100mcg\ml 545
3. 100mcg\ml 548
4. 100mcg\ml 549
5. 100mcg\ml 546

Test concentration of Chlorhexidine Gluconate used = 100mcg\ml.
544+545+548+549+546
Average area = ----------------------------------- = 546.4
5
(544-546.4)2 +(545-546.4)2 +(548-546.4)2 +(549-546.4)2 +(546-546.4)2

Dev. S =  --------------------------------------------------------------------------------------- 1/2
5-1
5.76+2.005+3.2 +1.99 +0.8

= ---------------------------------------1/2
4
13.755
=  ----- ----1/2
4
=  3.439  1/2
= 1.854
1.854 x 100
RSD = ------------------ = 0.339 %
546.4

RUGGEDNESS STUDY
The ruggedness of assay method of Chlorhexidine Gluconate in Chlortex Mouthwash
shall be studied by analyzing the samples from same lot by two different analysts from same

Wt. of Wt. of Measure Measured % of Wt. of Wt. of Measured Measur % of
Ref. Sample d Absor- Chlorhexi Ref. Sample Absor- ed Chlorhexid
Standard ml Absor- bance at dine Standard ml bance at Absor- ine
gm. bance 410nm of Gluconate gm. of Ref. bance at Gluconate
at sample Std. 410nm
of Ref. of
Std. sample
01 0.4998 50ml 544 545 100.49 0.5090 50ml 548 547 99.99
02 0.4989 50ml 546 542 99.04 0.4992 50ml 542 545 100.39
03 0.5012 50ml 543 545 99.69 0.4984 50ml 542 545 100.23
Label Claim 0.2% Chlorhexidine Label Claim 0.2% Chlorhexidine
Gluconate Gluconate
Mean 99.74% Mean 100.02%
Mean : 99.98 %

Name of the Product: BRONSYP SYRUP
assurance that the analytical method for assay of Salbutamol Sulphate,
Bromhexine Hcl & Guaiphensin in Bronsyp Syrup will give results with
acceptable Accuracy, Linearity, Repeatability, Relative Standard Deviation &
Ruggedness when applied to the Bronsyp Syrup.
of Salbutamol Sulphate, Bromhexine Hcl & Guaiphensin.
1. Linearity.
2. Repeatability.
4. Ruggedness.

METHOD :
(Salbutamol Sulphate)
1. Weigh accurately 100mg. of Salbutamol Sulphate as Reference standard and dissolve in
100 ml of water and prepare the dilution 100mcg/ml. in water.
2. Accurately weigh quantity of sample is extracted with water to get 100mcg./ml in

respect of Salbutamol Sulphate.
3. Pipette 3ml, 4ml, 5ml, 6ml & 7ml of standard and sample solution and add 3ml of
Sodium Carbonate solution, 0.2ml of P-aminophenol reagent and volume is adjusted to
10ml with water.
5. Plot linearity curve from the different concentration corresponding area of Salbutamol
Sulphate peak. Taking concentration on X-axis and corresponding peak on Y-axis. Plot
graph should be connected at least three points.
6. Plot repeatability curve from by taking Salbutamol Sulphate solution serial no. on X-
axis and observed peak area on Y-axis.

LINEARITY STUDY
Linearity of assay method of Salbutamol Sulphate in Bronsyp Syrup shall be studied by

measuring the absorbance of the standard Salbutamol Sulphate in solution of 30 – 70mcg/ml
concentration.
Sr.No. Concentration of Salbutamol Sulphate in mcg\ml Peak area of
1. 30mcg\ml 695
2. 40mcg\ml 739
3. 50mcg\ml 795
4. 60mcg\ml 829
5. 70mcg\ml 888

REPEATIBILITY STUDY
Repeatibility of assay method of Salbutamol Sulphate in Bronsyp Syrup shall be

studied by measuring the absorbance of the standard Salbutamol Sulphate in solution of 30
mcg/ml concentration & range 635nm. Repeatibility curve shall be plotted for absorbance

1 30mcg\ml 698
2. 30mcg\ml 700
3. 30mcg\ml 697
4. 30mcg\ml 695
5. 30mcg\ml 703

Test concentration of Salbutamol Sulphate used = 30mcg\ml.
698+700+697+695+703
Average area = ----------------------------------- = 698.6
5
(698-698.6)2 +(700-698.6)2 +(697-698.6)2 +(695-698.6)2 +(703-698.6)2

Dev. S =  --------------------------------------------------------------------------------------- 1/2
5-1
0.36+ 1.96+ 2.56+ 12.96 + 19.36

= -------------------------------------------1/2
4
37.2
=  ----- ----1/2
4
=  9.3  1/2
= 3.049
3.049 x 100
RSD = ------------------ = 0.436 %
698.6

RUGGEDNESS STUDY
The ruggedness of assay method of Salbutamol Sulphate in Bronsyp Syrup shall be

Ref. Sample d Absor- Salbutam Ref. Sample Absor- Absor- Salbutam
Standard ml Absor- bance at ol Standard ml bance at bance at ol
of Ref.
Std.
01 0.1209 5ml 698 702 100.32 0.1218 5ml 695 694 100.35
02 0.1216 5ml 694 697 100.76 0.1206 5ml 694 694 99.22
03 0.1206 5ml 695 698 99.77 0.1218 5ml 698 701 100.93
Label Claim 2mg. / 5ml Label Claim 2 mg. / 5ml
Mean 100.28% Mean 100.93%
Mean : 100.16%

METHOD :
(Bromhexine Hcl)
1. Weigh accurately 40mg. of Bromhexine Hcl. and transfer to a separating funnel, basify
with 5ml of dilute Ammonia solution , add 25ml. of water and extract with for 25ml
portions of Chloroform layer through anhydrous Sodium Sulphate previously washed
with Chloroform. Evaporate Chloroform layer and dissolve residue in 0.1M Methanolic
Hydrochloric Acid to produce 100ml. (400mcg./ ml.). Further make the dilution with
Methanolic Hydrochloric Acid to get final concentration of 40mcg/ml.
2. Measure accurately sample equivalent to 4mg. of Bromhexine Hcl., add 25ml. water
and continue with the procedure as described under preparation of standard solution.
3. Pipette 2ml, 3ml, 4ml, 5ml & 6ml of standard and sample solution, add 8ml of
P-dimethyl-aminobenzaldehyde (10%w/v in Methanol) & 5ml. of Hydrochloric Acid
mix and allow the stand for 10 minutes and then make up the volume to 25ml. with
water.
4. Measure the absorbance at maximum at about 425nm, against respective blank and
5. Plot linearity curve from the different concentration corresponding area of Bromhexine
Hcl peak. Taking concentration on X-axis and corresponding peak on Y-axis. Plot
6. Plot repeatability curve from by taking Bromhexine Hcl. solution serial no. on X-axis
and observed peak area on Y-axis.

LINEARITY STUDY
Linearity of assay method of Bromhexine Hcl in Bronsyp Syrup shall be studied by measuring
the absorbance of the standard Bromhexine Hcl in solution of 3.2 – 9.6mcg/ml concentration &
Sr.No. Concentration of Bromhexine Hcl. in mcg\ml Peak area of
1. 3.2mcg\ml 545
2. 4.8mcg\ml 598
3. 6.4mcg\ml 642
4. 8.0mcg\ml 691
5. 9.6mcg\ml 732

REPEATIBILITY STUDY
Repeatibility of assay method of Bromhexine Hcl in Bronsyp Syrup shall be studied by
measuring the absorbance of the standard Bromhexine Hcl in solution of 3.2mcg/ml
concentration.
Sr. No. Concentration in mcg\ml Peak area

1 3.2mcg\ml 545
2. 3.2mcg\ml 547
3. 3.2mcg\ml 548
4. 3.2mcg\ml 545
5. 3.2mcg\ml 542


Test concentration of Bromhexine Hcl. used = 3.2mcg\ml.
545+547+548+545+542
Average area = ----------------------------------- = 545.4
5
(545-545.4)2 +(547-545.4 )2 +(548-545.4)2 +(545-545.4)2 +(542-545.4)2

Dev. S =  --------------------------------------------------------------------------------------- 1/2
5-1
0.16+2.56+6.76+0.16+11.56
= --------------------------------------------1/2
4
21.2
=  ----- ----1/2
4
=  5.3  1/2
= 2.302
2.302 x 100
RSD = ------------------ = 0.422 %
545.4

RUGGEDNESS STUDY
The ruggedness of assay method of Bromhexine Hcl in Bronsyp Syrup shall be

Ref. Sample d Absor- Bromhex Ref. Sample Absor- Absor- Bromhexi
Standard ml Absor- bance at ine Standard ml bance at bance at ne
of Ref.
Std.
01 0.0398 5ml 548 546 99.13 0.0396 5ml 545 542 98.45
02 0.0396 5ml 544 548 99.72 0.0399 5ml 541 543 100.11
03 0.0399 5ml 543 545 100.11 0.0401 5ml 542 543 100.43
Label Claim 4mg. Bromhexine/5ml Label Claim 4mg. Bromhexine/5ml
Mean 99.65% Mean 99.66%
Mean : 99.66

METHOD :
(Guaiphenesin)
1. Weigh accurately 100mg. of Guaiphenesin, transfer to a separating funnel. Add 20ml of

water and 5ml. of Sodium hydroxide solution (20%w/v). Extract with for 40ml portions
of petroleum ether (60-80oC) . Discard ether layer, collect the aqueous layer and extract
for 20ml portions of chloroform. Pass combined chloroform layer throught anhydrous
sodium Sulphate and make up to 100ml with chloroform.
2. Measure accurately sample equivalent to 100mg. of substance, add 20ml of water and
continue the procedure as described under preparation of standard solution.
3. Pipette 5ml, 6ml, 7ml, 8ml & 10ml of standard and sample solution, and make
up the volume 100ml. with methanol.
4. Measure the absorbance at maximum at about 275nm, against respective blank and

Guaiphenesin peak. Taking concentration on X-axis and corresponding peak on Y-axis.
Plot graph should be connected at least three points.
6. Plot repeatability curve from by taking Guaiphenesin solution serial no. on X-axis and

LINEARITY STUDY
Linearity of assay method of Guaiphenesin in Bronsyp Syrup shall be studied by measuring the
absorbance of the standard Guaiphenesin in solution of 50 – 100mcg/ml concentration & range
Sr.No. Concentration of Guaiphenesin in mcg\ml Peak area of

1. 50mcg\ml 419
2. 60mcg\ml 460
3. 70mcg\ml 502
4. 80mcg\ml 542
5. 100mcg\ml 605

REPEATIBILITY STUDY
Repeatibility of assay method of Guaiphenesin in Bronsyp Syrup shall be studied by measuring
the absorbance of the standard Guaiphenesin in solution of 50 mcg/ml concentration & range
275nm. Repeatibility curve shall be plotted for absorbance against concentration.

1 50mcg\ml 421
2. 50mcg\ml 419
3. 50mcg\ml 424
4. 50mcg\ml 422
5. 50mcg\ml 420


Test concentration of Guaiphensin used = 50mcg\ml.
421+419+424+422+420
Average area = ----------------------------------- = 421.2
5
(421-421.2)2 +(419-421.2)2 +(424-421.2)2 +(422-421.2)2 +(420-421.2)2

Dev. S =  --------------------------------------------------------------------------------------- 1/2
5-1
0.04+4.84+ 7.84 + 0.64 + 1.44

= --------------------------------------------1/2
4
14.8
=  ----- ----1/2
4
=  3.7  1/2
= 1.924
1.924 x 100
RSD = ------------------ = 0.457 %
421.2

RUGGEDNESS STUDY
The ruggedness of assay method of Guaiphensin in Bronsyp Syrup shall be studied by

Wt. of Wt. of Measure Measured % of Wt. of Ref. Wt. of Measured Measured % of
Ref. Sample d Absor- Guaiph Standard Sample Absor- Absor- Guaiphen
Standard ml Absor- bance at enesin gm. ml bance at bance at esin
gm. bance 275nm of of Ref. 275nm
of Ref.
Std.
01
02
03
Label Claim 50mg Guaiphenesin/5ml Label Claim 50mg Guaiphenesin/5ml
Mean Mean
Mean :

Name of the Product: BECLOSAR-NC CREAM
assurance that the analytical method for assay of Beclomethasone Dipropionate,
Neomycin Sulphate & Clotrimazole in Beclosar-NC Cream will give results
with acceptable Accuracy, Linearity, Repeatibility, Relative Standard Deviation
& Ruggedness when applied to the Beclosar-NC Cream.
of Beclomethasone Dipropionate, Neomycin Sulphate & Clotrimazole.
1. Linearity.
2. Repeatibility.
4. Ruggedness.

METHOD :
(Beclomethasone Dipropionate)
1. Weigh accurately 50mg. of Beclomethasone Dipropionate and dissolve in 100ml. of

methanol. Further dilute 1ml. and make up the volume to 50ml. with methanol. This
makes 10mcg./ml dilution of Beclomethasone Dipropionate.
2. Weight accurately sample (cream) equivalent to 0.5mg of Beclomethasone
Dipropionate and dissolve in 25ml. of methanol. Filter and then make up the volume to
50ml. with methanol.
3. Pipette 1ml, 2ml, 3ml, 4ml & 5ml of standard and sample solution, add 6ml. of 0.1M
tetrabutyl ammonium hydroxide (TBAH) reagent and 2ml. of 0.5%w/v of Tetrazolium
blue (BTZ) solution. Heat the solution on water bath for 15minutes, cool the solution
and add 10ml. Glacial Acetic acid.

Beclomethasone Dipropionate peak. Taking concentration on X-axis and
corresponding peak on Y-axis. Plot graph should be connected at least three points.
6. Plot repeatability curve from by taking Beclomethasone Dipropionate solution serial

no. on X-axis and observed peak area on Y-axis.

LINEARITY STUDY
Linearity of assay method of Beclomethasone Dipropionate in Beclosar-NC Cream shall be
studied by measuring the absorbance of the standard Beclomethasone Dipropionate in solution
of 1 – 5mcg/ml concentration & range 515nm. Linearity curve shall be plotted for absorbance
Sr.No. Concentration of Beclomethasone Dipropionate in mcg\ml Peak area of

1. 1mcg\ml 680
2. 2mcg\ml 742
3. 3mcg\ml 791
4. 4mcg\ml 839
5. 5mcg\ml 872

REPEATIBILITY STUDY
Repeatibility of assay method of Beclomethasone Dipropionate in Beclosar-NC Cream shall be
studied by measuring the absorbance of the standard Beclomethasone Dipropionate in solution
of 1 mcg/ml concentration & range 515nm. Repeatibility curve shall be plotted for absorbance

1 1mcg\ml 680
2. 1mcg\ml 682
3. 1mcg\ml 681
4. 1mcg\ml 680
5. 1mcg\ml 685

Test concentration of Beclomethasone Dipropionate used = 1mcg\ml.

680+682+681+680+685
Average area = ----------------------------------- = 681.6
5
(680-681.6)2 +(682-681.6)2 +(681-681.6)2 +(680-681.6)2 +(685-681.6)2

Dev. S =  --------------------------------------------------------------------------------------- 1/2
5-1
2.56+0.16+0.36+2.56+11.56
= ---------------------------------------1/2
4
17.2
=  ----- ----1/2
4
=  4.3  1/2
= 2.0736
2.0736 x 100
RSD = ------------------ = 0.3042 %
681.6

RUGGEDNESS STUDY

The ruggedness of assay method of Beclomethasone Dipropionate in Beclosar-NC
Cream shall be studied by analyzing the samples from same lot by two different analysts from
same laboratory or at two different laboratories by two different analysts on two different days.

Ref. Sample d Absor- Beclome Ref. Sample Absor- Absor- Beclomet
Standard gm Absor- bance at thasone Standard ml bance at bance at hasone
of Ref.
Std.
01 0.0498 1.9969 683 681 99.52 0.0506 1.9978 682 684 100.62
02 0.0492 2.0013 685 686 98.53 0.0494 1.9982 682 684 99.19
03 0.0502 1.9975 684 686 100.88 0.0497 2.0018 684 686 99.61
Label Claim 0.025% w/w Label Claim 0.025% w/w
Mean 99.64% Mean 100.14%
Mean : 99.89 %

METHOD :
(Neomycin Sulphate)
1. Weigh accurately 100mg. of Neomycin Sulphate and dissolve in 100ml. of phosphate

buffer pH 8.0 and prepare the dilution 100mcg/ml. in phosphate buffer.
2. Weight accurately sample (cream) equivalent to 10mg of Neomycin Sulphate, suspend

in 25ml. of phosphate buffer and extract with Chloroform (3 x 30ml). Collect
chloroform in a dry 100ml volumetric flask filtering each layer through same
anhydrous sodium Sulphate bed. Rinse sodium Sulphate layer with chloroform, the
combined chloroform layer is made up of volume. Aqueous buffered layer heated in
water bath till no perceptible smell of chloroform is diluted to 100ml with phosphate
buffer and used for estimation of Neomycin Sulphate.
3. Pipette 5ml, 6ml, 7ml, 8ml & 9ml of standard and sample solution, add 5ml. of freshly
prepared ninhydrin reagent and heat in water bath for 20 minutes, cool, add 2 ml. of
methanol and dilute to 50ml with phosphate buffer.
5. Plot linearity curve from the different concentration corresponding area of Neomycin
Sulphate peak. Taking concentration on X-axis and corresponding peak on Y-axis. Plot
6. Plot repeatability curve from by taking Neomycin Sulphate solution serial no. on X-
axis and observed peak area on Y-axis.

LINEARITY STUDY
Linearity of assay method of Neomycin Sulphate in Beclosar-NC Cream shall be studied by

measuring the absorbance of the standard Neomycin Sulphate in solution of 10 – 18mcg/ml
concentration.
Sr.No. Concentration of Neomycin Sulphate in mcg\ml Peak area of
1. 10mcg\ml 652
2. 12mcg\ml 698
3. 14mcg\ml 749
4. 16mcg\ml 805
5. 18mcg\ml 862

REPEATIBILITY STUDY
Repeatibility of assay method of Neomycin Sulphate in Beclosar-NC Cream shall be studied

by measuring the absorbance of the standard Neomycin Sulphate in solution of 10mcg/ml
concentration.

1 10mcg\ml 650
2. 10mcg\ml 652
3. 10mcg\ml 654
4. 10mcg\ml 648
5. 10mcg\ml 657

Test concentration of Neomycin Sulphate used = 10mcg\ml.

650+652+654+648+657
Average area = ----------------------------------- = 652.2
5
(650-652.2)2 +(652-652.2)2 +(654-652.2)2 +(648-652.2)2 +(657-652.2)2

Dev. S =  --------------------------------------------------------------------------------------- 1/2
5-1
4.84+0.04+3.24+17.64+23.04
= ---------------------------------------1/2
4
48.8
=  ----- ----1/2
4
=  12.2  1/2
= 3.493
3.493 x 100
RSD = ------------------ = 0.536 %
652.2

RUGGEDNESS STUDY

The ruggedness of assay method of Neomycin Sulphate in Beclosar-NC Cream shall
be studied by analyzing the samples from same lot by two different analysts from same

Ref. Sample d Absor- Neomyci Ref. Sample Absor- Absor- Neomyci
Standard gm. Absor- bance at n Standard gm. bance at bance at n
of Ref.
Std.
01 0.0998 2.0256 656 652 98.03 0.0995 2.0160 652 649 98.37
02 0.0995 2.0106 650 652 99.37 0.0998 2.0059 653 654 99.71
03 0.1008 1.9994 651 649 100.62 0.1029 1.9989 656 658 103.33
Mean 99.34% Mean 100.45 %
Mean : 99.89%
Standard Deviation : ±10

METHOD :
(Clotrimazole)
1. Weigh accurately reference standard 100mg. of Clotrimazole and dissolve in 100ml. of

0.1M hydrochloric acid and prepare the dilution 100mcg./ml of Clotrimazole.
2. Weigh accurately sample equivalent to 100mg. of Clotrimazole and dissolve in 100ml.
of 0.1M hydrochloric acid and prepare the dilution 100mcg./ml of Clotrimazole.
3. Pipette 1ml, 2ml, 3ml, 4ml & 5ml of standard and sample dilution, add 3ml. of citric
acid solution and 2ml of dye solution. Extract the colour with three 8 ml portions of
chloroform. Make up the volume to 25ml and pass through anhydrous sodium Sulphate.
5. Plot linearity curve from the different concentration corresponding area of Clotrimazole
6. Plot repeatability curve from by taking Clotrimazole solution serial no. on X-axis and

LINEARITY STUDY
Linearity of assay method of Clotrimazole in Beclosar-NC Cream shall be studied by

measuring the absorbance of the standard Clotrimazole in solution of 4 – 20mcg/ml
concentration.
Sr.No. Concentration of Clotrimazole in mcg\ml Peak area of
1. 4mcg\ml 595
2. 8mcg\ml 632
3. 12mcg\ml 682
4. 16mcg\ml 735
5. 20mcg\ml 795
REPEATIBILITY STUDY
Repeatibility of assay method of Clotrimazole in Beclosar-NC Cream shall be studied by

measuring the absorbance of the standard Clotrimazole in solution of 4mcg/ml concentration &

1 4mcg\ml 595
2. 4mcg\ml 592
3. 4mcg\ml 593
4. 4mcg\ml 595
5. 4mcg\ml 596
Test concentration of Neomycin Sulphate used = 4mcg\ml.
595+592+593+595+596
Average area = ----------------------------------- = 594.2
5
(595-594.2)2 +(592-594.2)2 +(593-594.2)2 +(595-594.2)2 +(596-594.2)2

Dev. S =  --------------------------------------------------------------------------------------- 1/2
5-1
0.64 + 4.84 + 1.44 + 0.64 + 3.24

= ------------------------------------------1/2
4
10.8
=  ----- ----1/2
4
=  2.7  1/2
= 1.6431
1.6431 x 100
RSD = ------------------ = 0.276 %
594.2

RUGGEDNESS STUDY

The ruggedness of assay method of Clotrimazole in Beclosar-NC Cream shall be

Wt. of Wt. of Measured Measur % of Wt. of Wt. of Measured Measured % of
Ref. Sample gm Absor- ed Clotrima Ref. Sample Absor- Absor- Clotrimaz
Standard bance at Absor- zole Standard gm bance at bance at ole
gm of Ref. bance at gm of Ref. 422nm
Std. 422nm Std. of sample
of
sample
01 0.0997 10.0056 596 598 100.03 0.1002 9.9969 593 595 100.57
02 0.1005 9.9969 592 593 100.76 0.0998 9.9982 598 595 99.32
03 0.9989 10.0125 593 590 99.32 0.1012 10.0015 597 599 101.53
Label Claim 1% w/w Label Claim 1% w/w
Mean 100.03% Mean 100.47%
Mean : 100.25%

Name of the Product: MOVSAR GEL
assurance that the analytical method for assay of Diclofenac Diethylamine &
Methylsalicylate in Movsar Gel will give results with acceptable Accuracy,
Linearity, Repeatibility, Relative Standard Deviation & Ruggedness when
applied to the Movsar Gel.
of Diclofenac diethylamine & Methylsalicylate.
1. Linearity.
2. Repeatibility.
4. Ruggedness.

METHOD :
(Diclofenac diethylamine )
1. Weigh accurately 50mg. of Diclofenac sodium working standard in a 100ml volumetric

flask. Dissolve with phosphate buffer and make up to 100ml with same solution and
prepared the dilution 50mcg./ml. of Diclofenac sodium.
2. Weight accurately sample equivalent to 5mg of Diclofenac sodium in a 100ml

volumetric flask and dissolve in 100ml phosphate buffer.
3. Pipette 10ml, 12ml, 15ml, 18ml & 20ml of standard and sample solution separately to
each funnel add 5ml of methylene blue solution and extract with (10+10+5ml) of
chloroform passing each extract through anhydrous sodium Sulphate, dilute to 25ml
with chloroform mix to make homogeneous.
5. Plot linearity curve from the different concentration corresponding area of Diclofenac
diethylamine peak. Taking concentration on X-axis and corresponding peak on Y-axis.
Plot graph should be connected at least three points.
6. Plot repeatability curve from by taking Diclofenac diethylamine solution serial no. on
X-axis and observed peak area on Y-axis.

LINEARITY STUDY
Linearity of assay method of Diclofenac diethylamine in Movsar Gel shall be studied by

measuring the absorbance of the standard Diclofenac diethylamine in solution of 20 –
40mcg/ml concentration & range 650nm. Linearity curve shall be plotted for absorbance
Sr.No. Concentration of Diclofenac diethylamine in mcg\ml Peak area of
1. 20mcg\ml 716
2. 24mcg\ml 742
3. 30mcg\ml 795
4. 36mcg\ml 842
5. 40mcg\ml 878

REPEATIBILITY STUDY
Repeatibility of assay method of Diclofenac diethylamine in Movsar Gel shall be studied by

measuring the absorbance of the standard Diclofenac diethylamine in solution of 20 mcg/ml
concentration.

1 20mcg\ml 719
2. 20mcg\ml 714
3. 20mcg\ml 718
4. 20mcg\ml 722
5. 20mcg\ml 720
Test concentration of Diclofenac diethylamine used = 20mcg\ml.
719+714+718+722+720
Average area = ----------------------------------- = 718.6
5
(719-718.6)2 +(714-718.6)2 +(718-718.6)2 +(722-718.6)2 +(720-718.6)2

Dev. S =  --------------------------------------------------------------------------------------- 1/2
5-1
0.16+21.16+ 0.36+ 11.56+ 1.96

= ------------------------------------------1/2
4
35.2
=  ----- ----1/2
4
=  8.8  1/2
= 2.9664
2.9664 x 100
RSD = ------------------ = 0.412 %
718.6

RUGGEDNESS STUDY

The ruggedness of assay method of Diclofenac diethylamine in Movsar Gel shall be

Ref. Sample Absorba Absorbanc Diclof Ref. Sample Absor- Absor- Diclofe
Standard gm. nce at e at Standard gm. bance at bance at
gm of Ref. 650nm of enac gm. of Ref. 650nm nac
Std. sample diethyl Std. of sample diethyl
amine amine
01 0.0493 0.5012 715 718 99.78 0.0498 0.4992 718 720 100.03
02 0.0501 0.5006 718 716 99.80 0.0494 0.4984 716 718 99.39
03 0.0499 0.4993 719 716 99.52 0.0502 0.5012 719 721 100.44
Mean 99.36% Mean 100.44
Mean : 99.88 %
METHOD :
(Methylsalicylate)
1. Weigh accurately 100mg. of Methylsalicylate as working standard in a 100ml

volumetric flask and dissolve in 0.02M hydrochloric acid and make up to produced
100ml, again dilute 15ml of the resulting solution to 100ml with 0.02M hydrochloric
acid.
2. Weight accurately equivalent to 100mg of Methyl salicylate in a 500ml conical flask

add 30ml of 0.5M sodium hydroxide and 20ml of methyl alcohol, mix and reflux for
one hour on steam bath to sponify and hydrolyse, cool to room temperature and add
20ml of 1.0M hydrochloric acid and dilute to 100ml with water and filter. Further dilute
15ml to 100ml with 0.02M hydrochloric acid.
3. Pipette 10ml, 15ml, 20ml, 25ml & 30ml of standard and sample solution in separately
funnel and extract with 25ml of chloroform, separate chloroform layer in a dry test
tube. To 5 ml of chloroform layer add 10ml of ferric ammonium sulfate reagent and
separate the aqueous layer.

Methylsalicylate peak. Taking concentration on X-axis and corresponding peak on Y-
axis. Plot graph should be connected at least three points.
6. Plot repeatability curve from by taking Methylsalicylate solution serial no. on X-axis
and observed peak area on Y-axis.

LINEARITY STUDY
Linearity of assay method of Methylsalicylate in Movsar Gel shall be studied by measuring the
absorbance of the standard Methylsalicylate in solution of 30 – 90mcg/ml concentration &
Sr.No. Concentration of Methylsalicylate in mcg\ml Peak area
1. 30mcg\ml 685
2. 45mcg\ml 752
3. 60mcg\ml 796
4. 75mcg\ml 855
5. 90mcg\ml 888

REPEATIBILITY STUDY
Repeatibility of assay method of Methylsalicylate in Movsar Gel shall be studied by

measuring the absorbance of the standard Methylsalicylate in solution of 30mcg/ml
concentration.

1 30mcg\ml 687
2. 30mcg\ml 685
3. 30mcg\ml 684
4. 30mcg\ml 686
5. 30mcg\ml 685

Test concentration of Methylsalicylate used = 30mcg\ml.
687+685+684+686+685
Average area = ----------------------------------- = 685.4
5
(687-685.4)2 +(685-685.4)2 +(684-685.4)2 +(686-685.4)2 +(685-685.4)2

Dev. S =  --------------------------------------------------------------------------------------- 1/2
5-1
2.56+0.16+1.96+0.36+0.16
= ----------------------------------------1/2
4
= 5.2
[ ----- ----1/2
4
=  1.3  1/2
= 1.1401
1.1401 x 100
RSD = ------------------ = 0.166 %
685.4

RUGGEDNESS STUDY

The ruggedness of assay method of Methylsalicylate in Movsar Gel shall be studied

Ref. Sample d Absorbance Methylsa Ref. Sample Absorbanc Absorban Methylsal
Standard gm Absorb at 530nm licylate Standard gm. e at ce at icylate
gm ance at of sample gm of Ref. 530nm
of Ref. Std. of sample
Std.
01 0.0998 0.9994 684 686 100.15 0.1018 0.9996 687 686 101.68
02 0.1009 0.9989 684 685 100.87 0.0998 1.0015 684 686 99.93
03 0.0998 1.0012 687 685 99.39 0.1009 0.9994 684 682 100.66
Label Claim 10% w/w Label Claim 10% w/w
Mean 100.31 % Mean 100.75%
Mean : 100.53%

Sarthak Biotech Pvt. LTD.: Assay Method Validation Name of The Product: Sarcip-500 Tablet

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Sarthak Biotech Pvt. Ltd.

Total Page: 01-06

Date of Issue/Effective Date : 18/11/2007 Review Date : 17/11/2009 Revision No. : 00

RESPONSIBILITY: 1. Quality Control Manager.

2. Asstt. Quality Control Chemist.

3. Relative Standard Deviation.

1. Weight accurately equivalent to about 100mg of Ciprofloxacin working Reference

3. Pipette 3ml,5ml,7ml,9ml and 11ml of standard and sample solution separately in a

5. Measure the absorbance at maximum at about 438nm,against water as blank and

6. Plot linearity curve from the different concentration corresponding area of

Linearity of assay method of Ciprofloxacin in Sarcip-500 Tables shall be studied by measuring

OBSERVATION TABLE FOR LINEARITY

Sr.No. Concentration of Ciprofloxacin in mcg\ml Peak area

Date of Issue/Effective Date : 18/11/2007 Review Date : 17/11/2009 Revision No. : 00

Repeatibility of assay method of Ciprofloxacin in Sarcip-500 Tables shall be studied by

OBSERVATION TABLE FOR REPEATIBILITY

Sr. No. Concentration in mcg\ml Peak area

Sarthak Biotech Pvt. Ltd. Total Page: 05-06

RELATIVE STANDARD DEVIATION

Test concentration of Ciprofloxacin used = 100mcg\ml.

Number of test solution used = 5

(604-604.8)2 +(608-604.8)2 +(603-604.8)2 +(605-604.8)2 +(604-604.8)2

Limit : ( Not more than 2.0% )

The ruggedness of an analytical method is the degree of reproducibility of the test

RUGGEDNESS STUDY DATA

Sr. Analyst- I (Rajnish Kohli) Analyst- II (Satpal Saini)

RESPONSIBILITY: 1. Quality Control Manager.

2. Asstt. Quality Control Chemist.

3. Relative Standard Deviation.

1. Weigh accurately equivalent to about 50mg of Ofloxacin working Reference

2. Pipette 3ml,4ml,5ml,6ml and 7ml of standard and sample solution separately in a

5. Measure the absorbance at maximum at about 437nm,against water as blank and

OBSERVATION TABLE FOR LINEARITY

Sr.No. Concentration of Ofloxacin in mcg\ml Peak area

Date of Issue/Effective Date : 18/11/2007 Review Date : 17/11/2009 Revision No. : 00

Repeatibility of assay method of Ofloxacin in Sarflox-200 Tables shall be studied by

OBSERVATION TABLE FOR REPEATIBILITY

S.No. Concentration in mcg\ml Peak area

Sarthak Biotech Pvt. Ltd. Total Page: 05-06

RELATIVE STANDARD DEVIATION

Test concentration of Ofloxacin used = 50mcg\ml.

Number of test solution used = 5

515 + 512 + 515 + 509 + 519

(515-513.4)2 +(512-513.4)2 +(515-513.4)2 +(509-513.4)2 +(519-513.4)2 1/2

Limit : ( Not more than 2.0% )

Sarthak Biotech Pvt. Ltd. Total Page: 06-06

The ruggedness of an analytical method is the degree of reproducibility of the test

RUGGEDNESS STUDY DATA

Sr. Analyst- I (Satpal Saini) Analyst- II (Rajnish Kohli)

Sarthak Biotech Pvt. Ltd. Total Page: 01-11

RESPONSIBILITY : 1. Quality Control Manager.

2. Asstt. Quality Control Chemist.

3. Relative Standard Deviation.

Sarthak Biotech Pvt. Ltd. Total Page: 02-11

1. Weight accurately equivalent to about 60mg of Paracetamol working Reference

2. Pipette 2ml,3ml,4ml,5ml and 6ml of standard and sample solution separately in a

Sarthak Biotech Pvt. Ltd. Total Page: 03-11

OBSERVATION TABLE FOR LINEARITY

Sr.No. Concentration of Paracetamol in mcg\ml Peak area

Sarthak Biotech Pvt. Ltd. Total Page: 04-11

Repeatibility of assay method of Paracetamol in Sarnim-P Tables shall be studied by measuring

OBSERVATION TABLE FOR REPEATBILITY