BIOWAIVER STUDY

Of
RIVO 100 mg ENTERIC COATED TABLETS.
(Acetylsalicylic acid 100 mg)
The Arab Drug Company for Pharmaceutical & Chemical
Industries.5-El massanei st. Al Amyria,Cairo-A.R.E.

VERSUS

.ASPRIN® PROTECT 100 mg GASTRORESISTANT TABLETS

(Acetylsalicylic acid 100 mg)
Bayer Britterfeld GmbH-Germany for Bayer Consumer Care
AG-Swizerland , packed by Memphis Co. for pharmaceutical and
Chemicals Industries – Egypt.
1.1 Introduction 1

[Link] for dissolution submission 3

[Link] 4
[Link] of 5
5. Product Information 5
[Link] Determination 8
[Link] methodology 8
[Link] method validation in term of calibration 10
6.4.2. Validation parameters 10
[Link]. Linearity 10
[Link]. Accuracy 11
[Link]. Precision 12
[Link]. Ruggedness 14
[Link]. Robustness 15
Validation collective Table 16
Validation conclusion 16
[Link] uniformity of test product dosage unit 17
[Link] testing on 12 dosage units 20
[Link] media used 22
8.2.1. pH 1.2 22
8.2.2. pH 4.5 23
8.2.3. pH 6.8 24
[Link].Dissolution at buffer stage 33
8.3. Equation & results for the 12 dosage units for all pH 42
[Link]& Equation at pH 6.8 43
[Link]& Equation at buffer stage 42
[Link] factor “f2” calculation for each pH 44
[Link] factor “f1” calculation for each pH 44
[Link] dissolution profile for all pH 45
[Link] method validation 47

1.1

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 Dissolution evaluation of two submitted (Generic & Innovator) Enteric Coated Tablets formulations
containing Acetylsalicylic acid has been carried out in the frame of a comparative study, to determine
the release of Acetylsalicylic acid from Rivo 100 mg Enteric Coated Tablets (Generic), a product of
The Arab Drug Company for Pharmaceutical & Chemical and Asprin® protect 100 mg Gastroresistant
tablets (Innovator), manufactured by Bayer Britterfeld GmbH-Germany for Bayer Consumer Care AG-
Swizerland ,packed by Memphis Co., for pharmaceutical and Chemicals Industries – Egypt.

Test (Generic) Drug: Rivo 100 mg Enteric Coated Tablets

1. Name & Dosage Form Rivo 100 mg Enteric Coated Tablets.

2
1. Name of active ingredient(s) Acetylsalicylic acid 100 mg
3 & conc.
1. Manufacturer The Arab Drug Company for Pharmaceutical & Chemical
4 Industries.5-El massanei st. Al Amyria,Cairo-A.R.E.

Reference (Innovator) drug: Asprin® protect 100 mg Gastroresistant tablets.

1. Name & Dosage Form Asprin® protect 100 mg Gastroresistant tablets.

5
1. Name of active ingredient(s) Acetylsalicylic acid 100 mg
6 & conc.
1. Manufacturer & country of Bayer Britterfeld GmbH-Germany for Bayer Consumer Care
7 origin AG-Swizerland ,packed by Memphis Co., for pharmaceutical
and Chemicals Industries – Egypt.

1.8. Name and address of Bioequivalence Center

Egyptian Research And Development Company , 18 Adly street –Down Town ,Cairo ,Egypt.

1.9 Affiliation Name Signature Date

1.9.1 Chairman of the board Dr. Fathy Ibrahim
1.9.2 Center manager Dr. Rasha Altahawy
1.9.3 Technical manager [Link] Darwish
1.9.4 * Chief analyst [Link] Wafy el-Din
* [Link] Manager [Link] Wahba
1.9.5 Quality assurance manager Dr. Noha Mohamed

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2. Reason for dissolution submission (CAPA approval is submitted)
2.1 Bio-waiver of one strength based on
approved bioequivalence study of -
other strength
2.2 Bio-waived active ingredient Acetylsalicylic acid 100 mg
2.3 Variation in : -
2.3.1 Change in inactive ingredients -
2.3.2 Change in raw materials' suppliers -
2.4 Re-registration -

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3.

3.1 Test product

Rivo 100 mg Enteric Coated Tablets.
3.1.1 Trade name Rivo
3.1.2 Dosage form Enteric Coated Tablets
3.1.3 Strength 100 mg.
3.1.4 Manufacturer,sponsor The Arab Drug Company for Pharmaceutical & Chemical Industries.5-El
massanei st. Al Amyria,Cairo-A.R.E.
3.1.5 Batch number 410599
3.1.6 Manufacture date 12/2014.
Expiry date 12/2016.
3.2 Reference product :
Asprin® protect 100 mg
3.2.1 Trade name Asprin protect
3.2.2 Dosage form Gastroresistant tablets.
3.2.3 Strength 100 mg
3.2.4 Manufacturer & sponsorBayer Britterfeld GmbH-Germany for Bayer Consumer Care AG-Swizerland
& country of origin ,packed by Memphis Co. for pharmaceutical and Chemicals Industries –
Egypt.

3.2.5 Batch number BTAE241 M

3.2.6 Manufacture date 4/2013
expiry
Expirydate
Date 4/2016
3.3 Conclusion (similarity factor "f2") for all pH
Standard media Similarity factor (f2) for Acetylsalicylic acid
Acid Stage -
Buffer Stage 92.06
pH 1.2 -
pH 4.5 -
pH 6.8 70.74
Center Manager Technical Manager

Dr/ Rasha Altahawy [Link]./ Wagih A. Darwish

Reviewed by Lab Technical manager
Dr. Dina Wahba Issue Date: 12/03/2015

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4. Dates of:
4.1 Contract with sponsor 14/02/2015
4.2 Start of analysis 10/02/2015
4.3 End of analysis 28/03/2015
4.4 Report issue 28/03/2015

5. Product Information
1. Item Test Product Reference Product
2. Product name Rivo 100 mg Enteric Coated Asprin® protect 100 mg
3. API(S) Tablets. acid
Acetylsalicylic Acetylsalicylic acid
4. Molecular formula C9H8O4 C9H8O4
& structure

Molecular mass 180.157 g/mol

Systematic (IUPAC) 2-(acetoxy)benzoic acid
name
CAS number 50-78-2 Yes
Drug Bank DB00945
5. Dosage form Enteric Coated Tablets Gastroresistant tablets.
6. Type of the product Tablets Tablets
7. (Immediate
Dosage or modified
regimen For long term use,is 1-2 tablets once For long term use,is 1-2 tablets
[Link] some circumstances ahigher once [Link] some circumstances
dose may be appropriate,especially ahigher dose may be
in short term,and up to 300 mg a day appropriate,especially in short
may be used on the advice of a term,and up to 300 mg a day may
doctor . be used on the advice of a doctor .
8. Strength 100 mg 100 mg
9. Batch number 410599 BTAE241 M
10. Expiry date 12/2016 04/2016
11. Storage conditions Store at temperature ≤ 30 ºC Store at temperature ≤ 30 ºC

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 Composition Each tablet contains of
Active ingredients
Acetylsalicylic acid 100mg
B.P.2013
Active ingredients
Maise starch 8 mg
B.P.2013
Microcrystalline cellulose 12 mg
B.P.2013
(Avicel pH 101) -
Subcoat
Opadry 5 mg In
house spec
(Mixture of Hypermellose
2910(USP)
Titanium dioxide
(USP),Triacetin(USP)
Entric coat
-Acryl-EZE 20 mg
In house spec

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OBJECTIVE
Application of a Biowaiver study of Acetylsalicylic acid 100 mg in Rivo 100 mg Enteric Coated
Tablets.
SCOPE:
Biowaiver study of Acetylsalicylic acid 100 mg in Rivo 100 mg Enteric Coated Tablets.
(Generic Drug) - Against Acetylsalicylic acid 100 mg in Asprin® protect 100 mg

RESPONSIBILITY
Chief analyst is responsible for realizing (undergoing) the planned biowaiver study.
Lab Technical manager is responsible for designing and reviewing the whole study.
General Characteristics

The prototypical analgesic used in the treatment of mild to moderate pain. It has anti-
inflammatory and antipyretic properties and acts as an inhibitor of cyclooxygenase which results
in the inhibition of the biosynthesis of prostaglandins. Acetylsalicylic acid also inhibits platelet
aggregation and is used in the prevention of arterial and venous thrombosis. (From Martindale,
The Extra Pharmacopoeia, 30th ed, p5) Chemistry
IUPAC name 2-(acetoxy) benzoic acid

Molecular weight 180.157 g/mol

Description: white crysatalline powder, stable in dry air,in moist air it gradually hydrolysed to
salicylic and acetic acids
Solubility: slightly soluble in water , freely soluble in alchol.

Pharmacology/Pharmacokinetics

Mechanism of action
The analgesic, antipyretic, and anti-inflammatory effects of acetylsalicylic acid are due to actions by
both the acetyl and the salicylate portions of the intact molecule as well as by the active salicylate
metabolite. Acetylsalicylic acid directly and irreversibly inhibits the activity of both types of
cyclooxygenase (COX-1 and COX-2) to decrease the formation of precursors of prostaglandins and
thromboxanes from arachidonic acid. This makes acetylsalicylic acid different from other NSAIDS

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 (such as diclofenac and ibuprofen) which are reversible inhibitors. Salicylate may competitively
inhibit prostaglandin formation. Acetylsalicylic acid's antirheumatic (nonsteroidal anti-
inflammatory) actions are a result of its analgesic and anti-inflammatory mechanisms; the
therapeutic effects are not due to pituitary-adrenal stimulation. The platelet aggregation-inhibiting
effect of acetylsalicylic acid specifically involves the compound's ability to act as an acetyl donor to
cyclooxygenase; the nonacetylated salicylates have no clinically significant effect on platelet
aggregation. Irreversible acetylation renders cyclooxygenase inactive, thereby preventing the
formation of the aggregating agent thromboxane A2 in platelets. Since platelets lack the ability to
synthesize new proteins, the effects persist for the life of the exposed platelets (7-10 days).
Acetylsalicylic acid may also inhibit production of the platelet aggregation inhibitor, prostacyclin
(prostaglandin I2), by blood vessel endothelial cells; however, inhibition prostacyclin production is
not permanent as endothelial cells can produce more cyclooxygenase to replace the non-functional
enzyme.

Absorption
Absorption is generally rapid and complete following oral administration but may vary according to
specific salicylate used, dosage form, and other factors such as tablet dissolution rate and gastric or
intraluminal pH.
Indications
For use in the temporary relief of various forms of pain, inflammation associated with various
conditions (including rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus
erythematosus, osteoarthritis, and ankylosing spondylitis), and is also used to reduce the risk of
death and/or nonfatal myocardial infarction in patients with a previous infarction or unstable angina
pectoris.
Side effects
Oral, mouse: LD50 = 250 mg/kg; Oral, rabbit: LD50 = 1010 mg/kg; Oral, rat: LD50 = 200 mg/kg.
Effects of overdose include: tinnitus, abdominal pain, hypokalemia, hypoglycemia, pyrexia,
hyperventilation, dysrhythmia, hypotension, hallucination, renal failure, confusion, seizure, coma,
and death.

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6. POTENCY DETERMINATION OF
RIVO 100 mg ENTERIC COATED TABLETS
The Arab Drug Company for Pharmaceutical & Chemical Industries.5-El massanei st. Al (
).Amyria,Cairo-A.R.E
&
ASPRIN® PROTECT 100 mg
Bayer Britterfeld GmbH-Germany for Bayer Consumer Care AG-Swizerland ,packed by Memphis
.[Link] pharmaceutical and Chemicals Industries – Egypt
6.1 ASSAY METHODOLOGY

SAMPLE PREPARATION: Twenty Tablets of Rivo 100 mg were weighed & grinded, an
accurate weight equivalent to the average weight of one tablet was transferred into 100-ml
volumetric flask, and 60 ml of diluting solution were added & sonicated for 30 minutes. The
tablets extract was cooled; if necessary then the volume was completed with same solvent,
homogenized and filtered through Millipore filter (0.45 µm). 1 ml was accurately transferred into
10-ml volumetric flask and volume completed with diluting solution. The procedure was repeated
. using twenty Asprin® protect 100 mg

STANDARD SOLUTION: An accurately weighed 100 mg Acetylsalicylic acid were

transferred into a 100-ml volumetric flask, and allowed to dissolve in 60-ml diluting solution. The
solution was sonicated for 30 minutes & volume was completed with the same solvent. 1 ml was
accurately transferred into 10-ml volumetric flask and volume was completed with diluting
.solution to obtain the final concentration of (100 µg/ml)
.DILUTING SOLUTION:Mixture of acetonitirile and formic acid (99:1)*

CHROMATOGRAPHIC CONDITIONS
Table1: Summarized HPLC-Separation Criteria
Column Inetrsil ODS-3 C18 (150 × 4.6mm, 5 µm)
Temperature 25°C
Phosphate buffer* (pH 2.2) : Acetonitrile
Mobile Phase
( 25 : 75 v/v )
Flow Rate 1.0 ml/min
Injection Volume 20 µl
Detector (UV) 280 nm , Ref off

2 4
* prepared by dissolving 7.8g NaH PO in 1L water and adjusting pH to 2.2 using phosphoric acid.

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 PROCEDURE FOR INJECTION
The HPLC method was adopted, 20 µl from each solution were injected into the HPLC-
.UV system. Peak areas were justified and quantified

CALCULATION
The percentages of Acetylsalicylic acid in Rivo 100 mg Enteric Coated Tablets or Asprin®
:protect 100 mg gastroresistant Tablets were calculated from the following equation
AT
.Percentage of Acetylsalicylic acid / Tablet = ----------- x 100
Ast
AT : Average area peak of test solution
Ast : Average area peak of standard solution

The amount of Acetylsalicylic acid per test Tablets (Rivo 100 mg [Link]) 104.63% (n = 5)*
The amount of Acetylsalicylic acid per reference Tablets (Asprin® protect 100 mg gastroresistant*
Tablets = 103.05 % (n = 3)
Acceptance range (90-110%) *

6.2 RESULTS & ACCEPTANCE VALUES

Table 2: Potency, Concentration and peak area

of Acetylsalicylic acid in Rivo 100 mg Enteric Coated Tablets
and Asprin® protect gastroresistant Tablets 100 mg

No. Peak area of Peak area of Peak area of

Standard Test Reference
1. 674.30 702.1 701.8
2. 676.20 708.1 702.1
3. 674.60 707.1 682.5
4. 679.40 703.1
.5 670.00 710.5
Average 674.9 706.2 695.5
RSD 0.50% 0.42% 1.61%
% of Acetylsalicylic acid 104.63% 103.05%

* Potency (104.63%) lies in the acceptance range (90 - 110%)

6.3 HPLC CHROMATOGRAMS Attached

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6.4 ASSAY METHOD VALIDATION IN TERM OF CALIBRATION Attached
CURVE
6.4.1. Introduction
[Link].Introductory

Validation of an analytical procedure is performed in order to demonstrate that the

procedure is suitable for its intended use. Validation is performed in order to show that the
result(s) generated by a particular analytical procedure are reliable and accurate. The principles
and practices of validation of analytical procedures are covered by the International Conference
of Harmonization (ICH).

6.4.2. Validation parameters

[Link]. Linearity
[Link].1Definition
The linearity of an analytical procedure is its ability (within a given range) to obtain test
results that are directly proportional to the concentration of analyte in the sample.

[Link].2 Demonstration
Linearity is usually demonstrated by the analysis of various concentrations of the
analyte(s) across the intended range, and represented graphically. A statistical analysis of the
data is usually required, such as the calculation of a regression line using the method of least
squares. A minimum of five concentrations is recommended.
[Link].3 Data of Linearity of Acetylsalicylic acid
Table 3: Average Peak Area Response for Different Concentrations of Acetylsalicylic acid
(25 – 200 µg/ml)
Acetylsalicylic
Acetylsalicylic *PAR
No. acid
acid (µg/ml)
(%)
1 25% 25 172.83
2 50% 50 350.86
3 75% 75 518.75
4 100% 100 701.23
5 125% 125 860.73
6 150% 150 1051.40
7 200% 200 1387.30
*PAR: Peak Area
Response

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 Figure1: Calibration Curve of Acetylsalicylic acid
Results & Statistics
Regression line equation Y = Bx ± A
Intercept A = 0.9861
Slope B (variable) = 6.9465
R² (correction coefficient) = 0.9998

Limit of Detection (LOD) = 3.3 x δ (standard deviation) / Slope = 0.5662µg/ml

Limit of Quantitation (LOQ) =10 x δ (standard deviation) / Slope = 1.7156 µg/ml

Comment: Linear relationship was obtained between the recorded peak areas and their
corresponding concentrations. The correlation coefficient was found to be 0.9998.

[Link]. Accuracy
[Link].1 Definition
The accuracy of an analytical procedure expresses the closeness of agreement between the
value which is accepted either as a conventional true value or an accepted reference value and the
value found. This is sometimes termed trueness.

[Link].2 Demonstration
Accuracy is usually demonstrated by adding known amounts of standard to the sample
matrix and determining the measured result using the analytical procedure. The recovery of
measured against actual amounts is then calculated. Usually a minimum of three determinations
at each of three concentrations across the intended range is recommended.

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 [Link].3 Data of Accuracy of Acetylsalicylic acid
Table 4: Acetylsalicylic acid Accuracy Data

Acetylsalicylic acid(µg/ml)
No.
Standard Total
Test Found Recovery (%)
Added (Theoretical)
1 83.68 40.00 123.68 125.08 101.13%
2 83.68 80.00 163.68 166.81 101.91%
3 83.68 120.00 203.68 206.40 101.33%
Mean Standard
101.46 % 0.40
Results & value: deviation:
Statistics Relative Standard
0.40%
Deviation
Comment: This study confirmed that the present method for determination of Acetylsalicylic
acid is specific and accurate.

[Link]. Precision
6.4 .2.3.1 Definition
The precision of an analytical procedure expresses the closeness of agreement (degree of
scatter) between a series of measurements obtained from multiple samplings of the same
homogeneous sample under prescribed conditions. Precision is usually expressed as the variance,
standard deviation or relative standard deviation of a series of measurements.

Repeatability expresses the precision under the same operating conditions over a
short interval of time. Repeatability is also termed intra-assay precision.

Precision should be considered at different levels as follows

6.4. [Link] Repeatability (Intra-assay precision)
6.4. [Link].1 Demonstration
Repeatability is usually demonstrated by repeated measurements of a single sample
(e.g. use of the analytical procedure within a laboratory over a short period of time using the
same analyst with the same equipment). A minimum of three determinations at each of three
concentrations across the intended range, or a minimum of six determinations at the test
concentration is recommended.

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 [Link].1.2 Inter Day Precision (Intermediate Precision)
Intermediate precision expresses within-laboratory variations: different days, different
analysts or equipment, etc.

6.4. [Link].1 Demonstration

Intermediate precision is usually demonstrated by repeated measurements of the
sample used in the repeatability experiment within the same laboratory. Usually the
repeatability experiment is repeated on the same sample by a different analyst, on a different
day or using different equipment if possible.

[Link].2. Intermediate Precision of Acetylsalicylic acid

Table 5: Precision Data & Statistical Analysis of Acetylsalicylic acid

Individual value of
Test Acetylsalicylic acid Individual value
(µg/ml) Parameters
No.
(1 day)
st
(2nd day) (1st day) (2nd day)
1 104.27 101.34 Mean: (X) 104.513 102.227
2 104.67 101.55 Variance 0.1470 0.4951
3 104.71 102.25 Standard Deviation 0.383 0.704%
4 104.08 102.86 Relative Standard 0.37% 0.69%
5 104.25 102.23 F-test (α =0.05)
3.367
6 105.11 103.13 F critical = 5.05

Comment: Six different samples were assayed in two different days. The results obtained were
compared statistically (F-test) and showed that no significant difference was obtained between
results of two days as shown in Table (5).

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 [Link]. Ruggedness

[Link].1 Definition
The Ruggedness of an analytical method is the degree of reproducibility of test results
obtained by the analysis of the same sample under variety of conditions, such as different
laboratories, different analysts, different instruments, different lots of reagents, different
elapsed assay time, different assay temperature, different days, etc.
Ruggedness is normally expressed as the lack of influence of test results of operational and
environmental variables of the analytical method.

Different Analysis

Analyst-to-analyst Sample Precision for Acetylsalicylic acid

The precision of the method is determined by assaying six replicates of homogeneous
sample of Acetylsalicylic acid to determine single analyst precision.
Another analyst make six replicates of the same sample are assayed by the method described
for the assay of Acetylsalicylic acid to provide information concerning analyst to analyst
precision.
The results are shown in the following table (6).

6.4. [Link] Demonstration

Intermediate precision is usually demonstrated by repeated measurements of the sample
used in the repeatability experiment within the same laboratory. Usually the repeatability
experiment is repeated on the same sample by a different analyst, on a different day, using
different equipment if possible

[Link].2 Data of Repeatability & Ruggedness Precision of Acetylsalicylic acid

Table 6: Acetylsalicylic acid Repeatability, Ruggedness& Statistical Analysis Precision Data
Individual value of
Test Acetylsalicylic acid Individual value
(µg/ml) Parameters
No.
(1 Analyst) (2nd Analyst)
st
(1st Analyst) (2nd Analyst)
1 104.27 101.90 Mean: (X) 104.513 101.757
2 104.67 102.64 Variance 0.1470 0.5143
3 104.71 101.09 Standard Deviation 0.383 0.717
4 104.08 101.89 Relative Standard 0.37% 0.70%
5 104.25 102.28 F-test (α =0.05)
3.498
6 105.11 100.74 F critical = 5.05

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Comment Six different samples were assayed by two different analysts; Results
obtained are reported in Tables (6). The obtained data were subjected to statistical
analysis (F-test) and showed that the assays performed by the two analysts have the
same degree of precision and are equivalent.

[Link]. Robustness
[Link].1 Definition
The Robustness of an analytical method is a measure of its capacity to remain unaffected by
small but deliberated variations in method parameters and provides an indication of its
reliability during normal usage. The robustness of the method is determined by assaying
three replicates of one homogeneous sample under varied conditions.

[Link].2 Variation in Column Temperature

Samples were assayed at three different Column Temperatures (20 oC, 25 oC & 30 oC).
The results obtained at the three flow rates conditions seemed to be equivalent as shown in
Table (7)
Table 7: Variation in Column Temperature for Acetylsalicylic acid
o
C 20 o
C 25 o
C 30
T1 693.4 676.9 679
Peak Area T2 692.2 679.6 674.9
T3 690.4 687.4 680
Mean 692.0 681.3 678.0
RSD 0.22 0.80 0.40
T1 2.938 2.933 2.931
Retention
T2 2.935 2.934 2.930
Time
T3 2.935 2.937 2.929
Mean 2.936 2.935 2.930
RSD 0.06 0.07 0.03
T1 1.07 1.08 1.07
Symmetry T2 1.05 1.06 1.07
T3 1.06 1.05 1.06
Mean 1.06 1.06 1.07
SD 0.01 0.02 0.01
RSDٌ 0.94 1.44 0.54

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 Validation Collective Table

Acceptance
No. Parameters Result
Criteria
Linearity
Acetylsalicylic R not less
1 Active R2 = 0.9998
acid than 0.999
Ingredient
Accuracy
Acetylsalicylic 100.00 ±
2 Active 101.13% - 101.91%
acid 3.00 %
Ingredient
F (test) , α= 0.05
Statistica

Analysis

Precision F calculated
3 F (test)
Active less than F
l

Ingredient calculated F(critcal) critical

=3.367 = 5.05
Ruggedness F (test) , α= 0.05
Statistical
Analysis

F calculated
Active F (test)
4 F(critcal) less than F
Ingredient calculated
= 5.05 critical
= 3.498
Acetylsalicylic Negligible difference in Negligible
5 Robustness
acid system suitability difference
Validation Conclusion
All the parameters considered in validation of item assay (Linearity, Accuracy, Precision,
Ruggedness & Robustness) are within the limits as reported in above table giving assurance that
the assay of Acetylsalicylic acid is suitable for its intended use. This method can be applied for
both routine analysis and biowaiver study of Acetylsalicylic acid.

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 7. CONTET UNIFORMITY OF TEST PRODUCT DOSAGE UNIT
"According to the USP"

Ten Tablets were weighed & grinded and the weights were documented. Transfer each tablets
content into 100 ml volumetric flask, add 60 ml diluting solution & sonicate for 30 minutes. Then,
cool if necessary, complete to volume with the same solvent, shake, dilute 1 ml to 10 ml with
diluting solution. The prepared samples were analyzed by applying the justified HPLC- UV
.method for the determination of Acetylsalicylic acid
.DILUTING SOLUTION:Mixture of acetonitirile and formic acid (99:1)*

PROCEDURE FOR INJECTION

From each Test 20 µl were injected to HPLC-UV system and separately record peak areas
for each sample preparation for Acetylsalicylic acid. Concentrations were calculated using the
following equation.

7.1 Description of method used

Chromatographic Conditions
Column Intersil ODS-3 C18 (150 × 4.6mm, 5 µm)
Temperature 25°C
Phosphate buffer* (pH 2.2) : Acetonitrile
Mobile Phase
( 25 : 75 v/v )
Flow Rate 1.0 ml/min
Injection Volume 20 µl
Detector (UV) 280 nm , Ref off

2 4
* prepared by dissolving 7.8 g NaH PO in 1L water and adjusting pH to 2.2 using phosphoric acid.

AT
Percentage of Acetylsalicylic acid / Tablet = ----------- x 100
Ast
AT : Average area peak of tested solution.
Ast : Average area peak of standard solution.

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7.2 Results & Acceptance values
Table 8: Content Uniformity testing of Acetylsalicylic acid
in Rivo 100 mg Enteric Coated Tablets.

No. Peak area Weight Peak area of Concentration %

of Standard Test
1 709.3 698.3 698.3 98.25 98.2
2 713.8 694.7 694.7 97.74 97.7
3 711.2 697.0 697.0 98.07 98.1
4 711.2 695.0 695.0 97.78 97.8
5 708.8 695.9 695.9 97.91 97.9
6 710.2 695.5 695.5 97.85 97.9
7 696.4 696.4 97.98 98.0
8 695.3 695.3 97.83 97.8
9 692.9 692.9 97.49 97.5
10 693.6 693.6 97.59 97.6
Average  710.8 695.46 97.85 97.85%
SD 1.78 1.58 0.22 0.22
RSD 0.25% 0.23% 0.23% 0.23%
* Average Content (97.85%) lies in the acceptance range (85 - 115%).

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According to USP30 the requirement for dosage uniformity are met if the acceptance value of the first
10 dosage units is less than or equal to L1%
Calculation of the acceptance value according to this formula for the first 10 Tablets:
_
| M - X | + KS
_
X Mean of individual contents expressed as a percentage of label claim
K Acceptability constant, If n=10, then K = 2.4
S Standard deviation
T assay %
M Reference value,
if T≤101.5%
_ _
if 98.5% ≤ X ≤ 101.5 %, then M = X
_
X < 98.5 %, then M = 98.5
_
X > 101.5 %, then M = 101.5
if T > 101.5%,
_ _
98.5% ≤ X ≤ T %, then M = X
_
X < 98.5 %, then M = 98.5
_
X > T, then M = T
Acceptance value = (98.5 – 97.85) + (2.4 x 0.22)
Acceptance value = 1.178 < L1 (15)
L1 The maximum allowed acceptance value = 15 unless otherwise specified in the individual
monograph.

CONCLUSION: Content uniformity within accepted criteria.

ERDC RIVO-ADCO-B004-03/15 Confidential Page 21 of 54

7.3 HPLC chromatograms (Attached)
8. Dissolution testing "on 12 dosage units"
8.1 Dissolution testing method (Reference attached)

8.1. EXPERIMENTAL
8.1.1 Apparatus:
- Sensitive balance, BOECO, BB1 31 UK. Preuse regular calibration was done daily.
- pH meter, WTW Wissenschaftlich-Technische Werkstätten GmbH, Germany.
- Dissolution tester SR8PLUS, Hanson Research Corporation, Chatsworth, CA 91311
USA.
- HPLC, Agilent series 1100, Agilent Technologies, Deutschland GmbH, Waldbronn,
Germany.
8.1.2 Reagents and Reference Standards:
- Acetylsalicylic acid , 99.32%, Supplied by Unipharma, Egypt.
- Sodium hydroxide, Fluka Analytical, Denmark.
- Potassium dihydrogen o-phosphate, Fisher Scientific, UK
- O-Phosphoric acid, Sigma Aldrich, USA
- Acetonitrile (HPLC -grade), Sigma Aldrich, USA
- Hydrochloric acid, Sigma Aldrich, USA
- Ultra pure filtered water (PURELAB Prima, VWS Ltd (ELGA-Bucks-UK)
- Sodium Acetate, Sigma Aldrich, USA
- Methanol (HPLC -grade), Sigma Aldrich, USA

8.1.3 HPLC Method of analysis:

[Link] Material and Reagent
Chromatographic conditions:
Column : Inertsil ODS-3 C18 (150 × 4.6mm, 5 µm)
Temperature : 25 oC
Flow rate : 1.0 ml/ minute (isocratic)
Detector : UV (280 nm)
Injection volume : 20 µl.
Mobile phase : Phosphate buffer* (pH 2.2): Acetonitrile (25: 75) v/v
2 4
* prepared by dissolving 7.8 g NaH PO in 1L water and adjusting pH to 2.2 using phosphoric acid

[Link] Preparation of Dissolution Media**:

- pH=1.2 is prepared by adding 250 ml 0.2 M potassium chloride to 425 ml 0.2 M HCl ,in and
volume was completed to 1000 ml with distilled water.
- pH= 4.5 is prepared by adding 2.99 g Sodium acetate trihydrate in 1000 ml volumetric flask , to
14 mL 2 N acetic acid solution ( prepared by dissolving 116 ml glacial acetic acid in 1000 ml
water) and dilute to 1000 mL with water.

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 - pH 6.8 is prepared as follows: Dissolve 0.05M monobasic potassium phosphate in distilled water
and adjust pH using 0.2M sodium hydroxide.
-Acid Stage: 1000 mL of 0.1 N hydrochloric acid
-Buffer Stage: 1000 mL of pH 6.8 phosphate buffer, prepared by mixing 0.1 N hydrochloric acid
with 0.20 M tribasic sodium phosphate (3:1) and adjusting, if necessary, with 2 N hydrochloric
acid or 2 N sodium hydroxide to a pH of 6.8 ±0.05
** According to USP (Buffer solutions)
[Link] Preparation of Calibration Curve for the Active Ingredient at three Different pH
Media (The most Suitable media (Acid stage& Buffer Stage),pH 1.2, 4.5, 6.8):

[Link].1 Stock Standard Solution for Acetylsalicylic acid:

- 100 mg of Acetylsalicylic acid standard was accurately weighed and transferred into 100
ml volumetric flask, 60 ml diluting solution was added to dissolve the Acetylsalicylic acid
working standard, sonicated for 30 minutes to obtain final concentration of 1000 µg /ml of
Acetylsalicylic acid .
- Serial dilutions were prepared using each dilution media and calibration curves were
constructed for each media.

8.1.4 Dissolution Study of Generic and Reference Drugs at Different pH- Values
(The most Suitable media (Acid stage& Buffer Stage),pH 1.2, 4.5, 6.8):

[Link] Dissolution conditions:

Apparatus : Apparatus I (Basket)
Rotation : 100 r.p.m
Sampling times : 15, 20, 30, 45, 60, 120 minutes for acid stage, 15, 20, 30, 45,
60,90 minutes for buffer stage
Dissolution medium : 1000 ml of each dissolution media (the most Suitable media
(Acid stage& Buffer Stage), pH (1.2, 4.5 &6.8) )
Temperature : 37 ± 0.5 ºC

[Link] Dissolution steps:

- Dissolution media (1000 ml) pH 1.2 was put separately in each one of the 12
vessels.
- 5 ml each of generic and reference products were put in each one of the 12 vessels.
- From each vessel 5 ml at sampling times was withdrawn, filtered using syringe filter
(0.45 µm, Pall Corporation, Michigan, USA) and replaced with another 5 ml of the
same dissolution media at 37 oC.
- The filtered samples were injected separately to HPLC system and peak areas were
justified and quantified according to the method of assay used.
- The percentage of dissolved Acetylsalicylic acid was calculated from its corresponding
regression equation after adopting the HPLC- procedure.
- Dissolution profile was constructed.
- Similarity factor (f2) = 50 × log (1 + 1/n∑ n (Rt – Tt ) 2 )- 0.5 ×100 was calculated

ERDC RIVO-ADCO-B004-03/15 Confidential Page 23 of 54

 - Similarity factor (f2) should be ≥ 50 %. All previously described stated steps were
repeated for each dissolution media at (The most Suitable media (Acid stage&
Buffer Stage),pH 1.2, 4.5&6.8)
-
8.2 Dissolution media used
8.2.1 pH 1.2

[Link] Dissolution of Acetylsalicylic acid at pH 1.2

Table 9: Data of Acetylsalicylic acid linearity (25 – 200 µg/ml) at pH 1.2

Average Peak Area Response for Different Concentrations of Acetylsalicylic acid
(25 – 200 µg/ml)

Conc. Peak Peak Peak Area Average

RSD
(µg/ml) Area 1 Area 2 3 Peak Area
25 199.9 199.2 199.1 199.4 0.2
50 336.0 335.2 336.0 335.7 0.1
75 478.4 478.5 479.1 478.7 0.1
100 651.2 653.5 673.4 659.4 1.9
150 965.1 964.65 964.0 964.6 0.1
200 1289.4 1293.2 1294.7 1292.4 0.2

Figure 2: The calibration curve of Acetylsalicylic acid at pH 1.2

[Link].1.1. Dissolution profile of Rivo 100 mg Enteric Coated Tablets against

Asprin® protect 100 mg in dissolution medium ( pH 1.2 )

***No peaks were detected for Acetylsalicylic acid at pH 1.2 as the dosage form is
enteric coated tablets. (Reference attached)

8.2.2 pH 4.5

ERDC RIVO-ADCO-B004-03/15 Confidential Page 24 of 54

 [Link] Dissolution of Acetylsalicylic acid at pH 4.5

Calibration curve of Acetylsalicylic acid at pH 4.5

Table 10: Data of Acetylsalicylic acid linearity (25 – 200 µg/ml) at pH 4.5
Average Peak Area Response for Different Concentrations of Acetylsalicylic acid
(25 – 200 µg/ml)

Conc. Peak Peak Peak Area Average

RSD
(µg/ml) Area 1 Area 2 3 Peak Area
25 185.2 185.6 185.5 185.4 0.1
50 349.1 349.2 348.9 349.1 0.0
75 522.5 522.1 521.2 521.9 0.1
100 697.5 696.6 697.7 697.3 0.1
150 1015.7 1015.4 1014.1 1015.1 0.1
200 1361.1 1361.5 1358.4 1360.3 0.1

Figure 3: The calibration curve of Acetylsalicylic acid at pH 4.5

Dissolution profile of Rivo 100 mg Enteric Coated Tablets against

Asprin® protect 100 mg in dissolution medium ( pH 4.5 )

***No peaks were detected for Acetylsalicylic acid at pH 4.5 as the dosage form is
enteric coated tablets. (Reference attached)

8.2.3 pH 6.8

ERDC RIVO-ADCO-B004-03/15 Confidential Page 25 of 54

 8.2.3.1Dissolution of Acetylsalicylic acid at pH 6.8
Calibration curve for Acetylsalicylic acid at pH 6.8

Table 11: Data of Acetylsalicylic acid at pH 6.8 (25- 200 µg/ml)

Average Peak Area Response for Different Concentrations of Acetylsalicylic acid
(25 – 200 µg/ml)

Conc. Peak Peak Peak Area Average RSD

(µg/ml) Area 1 Area 2 3 Peak Area
25 172.3 172.6 172.6 172.5 0.1
50 337.2 341.0 336.0 338.1 0.8
75 503.5 502.2 501.1 502.3 0.2
100 662.0 663.5 663.6 663.0 0.1
150 987.6 986.1 989.4 987.7 0.2
200 1307.8 1308 1310.2 1308.7 0.1

Figure 4: The calibration curve of Acetylsalicylic acid dissolved at pH 6.8

[Link] Dissolution profile of Rivo 100 mg Enteric Coated Tablets against

Asprin® protect 100 mg in dissolution medium (pH 6.8)

[Link].1 The results (percent of Acetylsalicylic acid dissolved in each time) of

ERDC RIVO-ADCO-B004-03/15 Confidential Page 26 of 54

 Rivo 100 mg Enteric Coated Tablets at pH 6.8.
Table 12: Results of dissolution for Test No.1 at pH 6.8
%
Time Conc
Peak Area Acetylsalicylic
(min) (µg/ml)
acid Released
15 401.2 59.79 59.79
20 561.3 84.47 84.47
30 660.1 99.70 99.70
45 693.3 104.82 104.82
60 710.9 107.53 107.53
90 725.9 109.84 109.84
Table 13: Results of dissolution for Test No.2 at pH 6.8
%
Time Conc
Peak Area Acetylsalicylic
(min) (µg/ml)
acid Released
15 381.8 56.80 56.80
20 550.4 82.79 82.79
30 682.4 103.14 103.14
45 720.3 108.98 108.98
60 722.3 109.29 109.29
90 722.2 109.27 109.27
Table 14: Results of dissolution for Test No.3 at pH 6.8
%
Time Conc
Peak Area Acetylsalicylic
(min) (µg/ml)
acid Released
15 448.3 67.05 67.05
20 613.7 92.55 92.55
30 666.6 100.70 100.70
45 713.1 107.87 107.87
60 723.2 109.43 109.43
90 725.7 109.81 109.81

Table 15: Results of dissolution for Test No.4 at pH 6.8

%
Time Conc
Peak Area Acetylsalicylic
(min) (µg/ml)
acid Released

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 15 409.1 61.01 61.01
20 529.7 79.60 79.60
30 531.4 79.86 79.86
45 677.1 102.32 102.32
60 691.3 104.51 104.51
90 705.6 106.71 106.71
Table 16: Results of dissolution for Test No.5 at pH 6.8
%
Time Conc
Peak Area Acetylsalicylic
(min) (µg/ml)
acid Released
15 433.5 64.77 64.77
20 609.8 91.95 91.95
30 690.3 104.36 104.36
45 715.4 108.23 108.23
60 712.1 107.72 107.72
90 708.0 107.08 107.08
Table 17: Results of dissolution for Test No.6 at pH 6.8
%
Time Conc
Peak Area Acetylsalicylic
(min) (µg/ml)
acid Released
15 430.1 64.25 64.25
20 585.8 88.25 88.25
30 634.7 95.79 95.79
45 672.3 101.58 101.58
60 682.5 103.15 103.15
90 689.4 104.22 104.22

Table 18: Results of dissolution for Test No.7 at pH 6.8

%
Time Conc
Peak Area Acetylsalicylic
(min) (µg/ml)
acid Released
15 399.9 59.59 59.59

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 20 534.8 80.39 80.39
30 645.2 97.40 97.40
45 626.2 94.48 94.48
60 637.9 96.28 96.28
90 660.6 99.78 99.78
Table 19: Results of dissolution for Test No.8 at pH 6.8
%
Time Conc
Peak Area Acetylsalicylic
(min) (µg/ml)
acid Released
15 344.3 51.02 51.02
20 496.3 74.45 74.45
30 614.6 92.69 92.69
45 663.8 100.27 100.27
60 661.9 99.98 99.98
90 661.0 99.84 99.84
Table 20: Results of dissolution for Test No.9 at pH 6.8
%
Time Conc
Peak Area Acetylsalicylic
(min) (µg/ml)
acid Released
15 402.0 59.92 59.92
20 553.7 83.30 83.30
45 597.8 90.10 90.10
60 639.7 96.56 96.56
30 642.1 96.93 96.93
90 654.5 98.84 98.84

Table 21: Results of dissolution for Test No.10 at pH 6.8

%
Time Conc
Peak Area Acetylsalicylic
(min) (µg/ml)
acid Released
15 413.2 61.64 61.64
20 550.0 82.73 82.73

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 30 630.8 95.18 95.18
45 611.5 92.21 92.21
60 624.1 94.15 94.15
90 636.6 96.08 96.08
Table 22: Results of dissolution for Test No.11 at pH 6.8
%
Time Conc
Peak Area Acetylsalicylic
(min) (µg/ml)
acid Released
15 389.2 57.94 57.94
20 551.0 82.88 82.88
30 621.2 93.70 93.70
45 644.0 97.22 97.22
60 642.5 96.99 96.99
90 638.7 96.40 96.40
Table 23: Results of dissolution for Test No.12 at pH 6.8
%
Time Conc
Peak Area Acetylsalicylic
(min) (µg/ml)
acid Released
15 388.7 57.87 57.87
20 530.1 79.66 79.66
30 571.7 86.07 86.07
45 605.3 91.25 91.25
60 613.6 92.53 92.53
90 620.1 93.54 93.54

[Link].2 The results (percent of Acetylsalicylic acid dissolved in each time) of

Asprin® protect 100 mg at pH 6.8.

Table 24: Results of dissolution for Reference No.1 at pH 6.8

Time Peak Conc % Acetylsalicylic
(min) Area (µg/ml) acid Released
15 471.9 70.69 70.69

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 20 520.0 78.10 78.10
30 517.3 77.69 77.69
45 718.9 108.77 108.77
60 714.3 108.06 108.06
90 718.4 108.69 108.69
Table 25: Results of dissolution for Reference No.2 at pH 6.8
Time Peak Conc % Acetylsalicylic
(min) Area (µg/ml) acid Released
15 348.0 51.59 51.59
20 487.4 73.08 73.08
30 640.4 96.66 96.66
45 723.6 109.49 109.49
60 721.0 109.09 109.09
90 715.2 108.19 108.19
Table 26: Results of dissolution for Reference No.3 at pH 6.8
Time Peak Conc % Acetylsalicylic
(min) Area (µg/ml) acid Released
15 383.5 57.06 57.06
20 561.6 84.52 84.52
30 696.5 105.31 105.31
45 709.5 107.32 107.32
60 707.3 106.98 106.98
90 697.9 105.53 105.53

Table 27: Results of dissolution for Reference No.4 at pH 6.8

Time Peak Conc % Acetylsalicylic
(min) Area (µg/ml) acid Released
15 448.5 67.08 67.08
20 501.8 75.30 75.30
30 666.1 100.63 100.63
45 693.0 104.77 104.77
60 688.6 104.09 104.09

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 90 684.7 103.49 103.49
Table 28: Results of dissolution for Reference No.5 at pH 6.8
Time Peak Conc % Acetylsalicylic
(min) Area (µg/ml) acid Released
15 283.2 41.60 41.60
20 431.0 64.39 64.39
30 650.7 98.25 98.25
45 692.1 104.63 104.63
60 690.7 104.42 104.42
90 689.0 104.16 104.16
Table 29: Results of dissolution for Reference No.6 at pH 6.8
Time Peak Conc % Acetylsalicylic
(min) Area (µg/ml) acid Released
15 389.1 57.93 57.93
20 565.0 85.04 85.04
30 674.8 101.97 101.97
45 685.6 103.63 103.63
60 683.0 103.23 103.23
90 677.1 102.32 102.32

Table 30: Results of dissolution for Reference No.7 at pH 6.8

Time Peak Conc % Acetylsalicylic
(min) Area (µg/ml) acid Released
15 420.6 62.78 62.78
20 558.0 83.96 83.96
30 677.5 102.38 102.38
45 656.1 99.08 99.08
60 653.2 98.64 98.64
90 650.4 98.21 98.21

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 Table 31: Results of dissolution for Reference No.8 at pH 6.8
Time Peak Conc % Acetylsalicylic
(min) Area (µg/ml) acid Released
15 307.7 45.38 45.38
20 440.9 65.91 65.91
30 575.7 86.69 86.69
45 654.5 98.84 98.84
60 649.7 98.10 98.10
90 643.3 97.11 97.11
Table 32: Results of dissolution for Reference No.9 at pH 6.8
Time Peak Conc % Acetylsalicylic
(min) Area (µg/ml) acid Released
15 342.3 50.71 50.71
20 510.8 76.69 76.69
30 631.7 95.32 95.32
45 644.3 97.27 97.27
60 641.0 96.76 96.76
90 634.5 95.75 95.75

Table 33: Results of dissolution for Reference No.10 at pH 6.8

Time Peak Conc % Acetylsalicylic
(min) Area (µg/ml) acid Released
15 403.8 60.19 60.19
20 547.5 82.34 82.34
30 601.3 90.64 90.64
45 630.0 95.06 95.06
60 623.7 94.09 94.09
90 619.3 93.41 93.41

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 Table 34: Results of dissolution for Reference No.11 at pH 6.8
Time Peak Conc % Acetylsalicylic
(min) Area (µg/ml) acid Released
15 254.7 37.21 37.21
20 513.4 77.09 77.09
30 589.9 88.88 88.88
45 626.2 94.48 94.48
60 624.3 94.18 94.18
90 628.7 94.86 94.86
Table 35: Results of dissolution for Reference No.12 at pH 6.8
Time Peak Conc % Acetylsalicylic
(min) Area (µg/ml) acid Released
15 349.8 51.87 51.87
20 509.2 76.44 76.44
30 607.9 91.65 91.65
45 624.6 94.23 94.23
60 620.0 93.52 93.52
90 613.6 92.53 92.53

8.2.4 The most suitable medium, USP reference method (Attached)

[Link] Dissolution of Acetylsalicylic acid at acid stage

Table 36: Data of Linearity of Acetylsalicylic acid (25 - 200 µg/ml) at acid stage
Average Peak Area Response for Different Concentrations of Acetylsalicylic acid
(25 – 200 µg/ml)

Conc. Peak Peak Peak Area Average

RSD
(µg/ml) Area 1 Area 2 3 Peak Area
25 146.8 147.5 148.1 147.5 0.4

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 50 276.9 276.3 276.4 276.5 0.1
75 404.4 403.7 403.5 403.9 0.1
100 540.2 539.8 538.3 539.4 0.2
150 804.4 803.2 804.4 804.0 0.1
200 1028.0 1036.0 1021.4 1028.5 0.7

Figure 5: The calibration curve of Acetylsalicylic acid at acid stage

[Link].1 Dissolution profile of Rivo 100 mg Enteric Coated Tablets against

Asprin® protect 100 mg in dissolution medium acid stage

***No peaks were detected for Acetylsalicylic acid at acid stage as the dosage form is
enteric coated tablets. (Reference attached)

8.2.4.2Dissolution of Acetylsalicylic acid at Buffer stage

Table 37: Data of Linearity of Acetylsalicylic acid (25 - 200 µg/ml) at Buffer stage
Average Peak Area Response for Different Concentrations of Acetylsalicylic acid
(25 – 200 µg/ml)

Conc. Peak Peak Peak Area Average RSD

(µg/ml) Area 1 Area 2 3 Peak Area
25 167.3 167.2 166.4 166.97 0.30
50 326.2 325.9 325.8 325.97 0.06
75 480.2 479.3 478.8 479.43 0.15

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 100 641.5 641.0 641.0 641.17 0.05
150 956.3 955.8 958.4 956.83 0.14
200 1267.1 1264.3 1264.0 1265.13 0.14

Figure 6: The calibration curve of Acetylsalicylic acid at Buffer stage

[Link] Dissolution profile of Rivo 100 mg Enteric Coated Tablets against

Asprin® protect 100 mg in dissolution medium Buffer Stage

[Link].1 The results (percent of Acetylsalicylic acid dissolved in each time) of

Rivo 100 mg Enteric Coated Tablets at Buffer Stage.

Table 38: Results of dissolution for Test No.1 at Buffer Stage

%
Time Conc
Peak Area Acetylsalicylic
(min) (µg/ml)
acid Released
15 330.1 50.81 50.81
20 429.1 66.56 66.56
30 465.2 72.31 72.31

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 45 518.7 80.82 80.82
60 514.4 80.13 80.13
90 515.3 80.28 80.28
Table 39: Results of dissolution for Test No.2 at Buffer Stage
%
Time Conc
Peak Area Acetylsalicylic
(min) (µg/ml)
acid Released
15 342.5 52.78 52.78
20 407.6 63.14 63.14
30 537.0 83.73 83.73
45 528.9 82.44 82.44
60 542.4 84.59 84.59
90 530.5 82.70 82.70
Table 40: Results of dissolution for Test No.3 at Buffer Stage
%
Time Conc
Peak Area Acetylsalicylic
(min) (µg/ml)
acid Released
15 313.0 48.09 48.09
20 422.1 65.45 65.45
30 544.7 84.96 84.96
45 542.3 84.57 84.57
60 540.0 84.21 84.21
90 527.1 82.15 82.15

Table 41: Results of dissolution for Test No.4 at Buffer Stage

%
Time Conc
Peak Area Acetylsalicylic
(min) (µg/ml)
acid Released
15 339.0 52.22 52.22
20 431.8 66.99 66.99
30 503.8 78.45 78.45
45 506.4 78.86 78.86
60 502.2 78.19 78.19
90 517.0 80.55 80.55
Table 42: Results of dissolution for Test No.5 at Buffer Stage
Time Peak Area Conc %

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 Acetylsalicylic
(min) (µg/ml)
acid Released
15 333.1 51.29 51.29
20 339.5 52.30 52.30
30 489.0 76.09 76.09
45 518.6 80.80 80.80
60 516.6 80.48 80.48
90 514.5 80.15 80.15
Table 43: Results of dissolution for Test No.6 at Buffer Stage
%
Time Conc
Peak Area Acetylsalicylic
(min) (µg/ml)
acid Released
15 310.5 47.69 47.69
20 415.8 64.44 64.44
30 532.7 83.05 83.05
45 539.5 84.13 84.13
60 529.1 82.47 82.47
90 521.8 81.31 81.31

Table 44: Results of dissolution for Test No.7 at Buffer Stage

%
Time Conc
Peak Area Acetylsalicylic
(min) (µg/ml)
acid Released
15 350.3 54.02 54.02
20 422.5 65.51 65.51
30 388.4 60.09 60.09
45 557.0 86.91 86.91
60 566.4 88.41 88.41
90 584.2 91.24 91.24
Table 45: Results of dissolution for Test No.8 at Buffer Stage
%
Time Conc
Peak Area Acetylsalicylic
(min) (µg/ml)
acid Released

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 15 303.7 46.61 46.61
20 436.8 67.79 67.79
30 538.4 83.95 83.95
45 576.1 89.95 89.95
60 578.3 90.30 90.30
90 574.5 89.70 89.70
Table 46: Results of dissolution forTest No.9 at Buffer Stage
%
Time Conc
Peak Area Acetylsalicylic
(min) (µg/ml)
acid Released
15 353.9 54.60 54.60
20 487.9 75.92 75.92
45 522.1 81.36 81.36
60 553.6 86.37 86.37
30 562.2 87.74 87.74
90 573.1 89.47 89.47

Table 47: Results of dissolution for Test No.10 at Buffer Stage

%
Time Conc
Peak Area Acetylsalicylic
(min) (µg/ml)
acid Released
15 332.8 51.24 51.24
20 443.7 68.88 68.88
30 450.3 69.93 69.93
45 543.8 84.81 84.81
60 553.0 86.28 86.28
90 562.0 87.71 87.71
Table 48: Results of dissolution for Test No.11 at Buffer Stage
%
Time Conc
Peak Area Acetylsalicylic
(min) (µg/ml)
acid Released
15 340.0 52.38 52.38

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 20 490.4 76.32 76.32
30 548.0 85.48 85.48
45 568.9 88.81 88.81
60 569.8 88.95 88.95
90 565.2 88.22 88.22
Table 49: Results of dissolution for Test No.12 at Buffer Stage
%
Time Conc
Peak Area Acetylsalicylic
(min) (µg/ml)
acid Released
15 345.1 53.20 53.20
20 471.1 73.24 73.24
30 505.2 78.67 78.67
45 534.9 83.40 83.40
60 541.6 84.46 84.46
90 547.9 85.46 85.46

[Link].2 The results (percent of Acetylsalicylic acid dissolved in each time) of

Asprin® protect 100 mg at Buffer Stage

Table 50: Results of dissolution for Reference No.1 at Buffer Stage

Time Peak Conc % Acetylsalicylic
(min) Area (µg/ml) acid Released
15 303.5 46.58 46.58
20 412.3 63.89 63.89
30 479.9 74.64 74.64
45 539.7 84.16 84.16
60 535.2 83.44 83.44
90 531.9 82.92 82.92
Table 51: Results of dissolution for Reference No.2 at Buffer Stage
Time Peak Conc % Acetylsalicylic
(min) Area (µg/ml) acid Released

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 15 325.6 50.09 50.09
20 419.0 64.95 64.95
30 513.6 80.01 80.01
45 513.6 80.01 80.01
60 506.4 78.86 78.86
90 514.2 80.10 80.10
Table 52: Results of dissolution for Reference No.3 at Buffer Stage
Time Peak Conc % Acetylsalicylic
(min) Area (µg/ml) acid Released
15 330.2 50.82 50.82
20 433.2 67.21 67.21
30 526.4 82.04 82.04
45 549.7 85.75 85.75
60 541.3 84.41 84.41
90 528.2 82.33 82.33

Table 53: Results of dissolution for Reference No.4 at Buffer Stage

Time Peak Conc % Acetylsalicylic
(min) Area (µg/ml) acid Released
15 350.6 54.07 54.07
20 482.7 75.09 75.09
30 518.2 80.74 80.74
45 509.6 79.37 79.37
60 521.6 81.28 81.28
90 515.9 80.37 80.37
Table 54: Results of dissolution for Reference No.5 at Buffer Stage
Time Peak Conc % Acetylsalicylic
(min) Area (µg/ml) acid Released
15 345.1 53.20 53.20
20 450.8 70.01 70.01
30 509.5 79.35 79.35
45 503.4 78.38 78.38

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 60 499.6 77.78 77.78
90 513.9 80.05 80.05
Table 55: Results of dissolution for Reference No.6 at Buffer Stage
Time Peak Conc % Acetylsalicylic
(min) Area (µg/ml) acid Released
15 329.8 50.76 50.76
20 411.7 63.79 63.79
30 515.4 80.29 80.29
45 552.0 86.12 86.12
60 544.3 84.89 84.89
90 538.3 83.94 83.94

Table 56: Results of dissolution for Reference No.7 at Buffer Stage

Time Peak Conc % Acetylsalicylic
(min) Area (µg/ml) acid Released
15 373.0 57.63 57.63
20 489.1 76.11 76.11
30 377.9 58.41 58.41
45 577.0 90.09 90.09
60 578.6 90.35 90.35
90 568.2 88.69 88.69
Table 57: Results of dissolution for Reference No.8 at Buffer Stage
Time Peak Conc % Acetylsalicylic
(min) Area (µg/ml) acid Released
15 269.4 41.15 41.15
20 393.9 60.96 60.96
30 514.3 80.12 80.12
45 576.1 89.95 89.95
60 568.1 88.68 88.68

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 90 565.9 88.33 88.33
Table 58: Results of dissolution for Reference No.9 at Buffer Stage
Time Peak Conc % Acetylsalicylic
(min) Area (µg/ml) acid Released
15 303.8 46.62 46.62
20 446.9 69.39 69.39
30 553.5 86.36 86.36
45 562.8 87.84 87.84
60 559.1 87.25 87.25
90 557.1 86.93 86.93

Table 59: Results of dissolution for Reference No.10 at Buffer Stage

Time Peak Conc % Acetylsalicylic
(min) Area (µg/ml) acid Released
15 358.1 55.26 55.26
20 482.1 74.99 74.99
30 528.9 82.44 82.44
45 555.3 86.64 86.64
60 544.9 84.99 84.99
90 539.4 84.11 84.11
Table 60: Results of dissolution for Reference No.11 at Buffer Stage
Time Peak Conc % Acetylsalicylic
(min) Area (µg/ml) acid Released
15 225.4 34.15 34.15
20 345.5 53.26 53.26
30 518.8 80.83 80.83
45 547.5 85.40 85.40
60 545.7 85.11 85.11
90 556.2 86.79 86.79

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 Table 61: Results of dissolution for Reference No.12 at Buffer Stage
Time Peak Conc % Acetylsalicylic
(min) Area (µg/ml) acid Released
15 309.2 47.48 47.48
20 446.0 69.25 69.25
30 531.8 82.90 82.90
45 553.7 86.39 86.39
60 548.3 85.53 85.53
90 540.0 84.21 84.21

8.3. Equations & results for the 12 dosage units for all pH

8.3.1. CALCULATIONS& EQUATION at pH 6.8.

AUCT- (13.3134) 1000 100
Acetylsalicylic acid (dissolved %) at pH 6.8 = ------------------ x -------- x -------
6.4873 Dosage 1000
(Test or ref.)
Where;
AUCT : Peak area of tested tablet
Slope : 6.4873
Intercept : 13.3134
Dosage of Test : one tablet equivalent to 100 mg Acetylsalicylic acid
Dosage of Reference: one tablet equivalent to 100 mg Acetylsalicylic acid

Table 62: Summary of percent released of Acetylsalicylic acid from Test at pH 6.8
Time
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 Mean SD RSD
(min)

15 59.79 56.80 67.05 61.01 64.77 64.25 59.59 51.02 59.92 61.64 57.94 57.87 60.14 13.15 6.98

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 20 84.47 82.79 92.55 79.60 91.95 88.25 80.39 74.45 83.30 82.73 82.88 79.66 83.58 18.33 6.24
30 99.70 103.14 100.70 79.86 104.36 95.79 97.40 92.69 90.10 95.18 93.70 86.07 94.89 19.24 7.48
45 104.82 108.98 107.87 102.32 108.23 101.58 94.48 100.27 96.56 92.21 97.22 91.25 100.48 16.50 6.19
60 107.53 109.14 109.43 104.51 107.72 103.15 96.28 99.98 96.93 94.15 96.99 92.53 101.53 12.93 6.04
90 109.87 109.23 109.81 106.71 107.08 104.22 99.78 99.84 98.84 96.08 96.40 93.54 102.62 6.64 5.74

Table 63: Summary of percent released of Acetylsalicylic acid from Reference at pH 6.8
Time
R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12 Mean SD RSD
(min)

15 70.69 51.59 57.06 67.08 41.60 57.93 62.78 45.38 50.71 60.19 37.21 51.87 54.51 14.60 18.50
20 78.10 73.08 84.52 75.30 64.39 85.04 83.96 65.91 76.69 82.34 77.09 76.44 76.91 17.05 8.77
30 77.69 96.66 105.31 100.63 98.25 101.97 102.38 86.69 95.32 90.64 88.88 91.65 94.67 19.47 8.36
45 108.77 109.49 107.32 104.77 104.63 103.63 99.08 98.84 97.27 95.06 94.48 94.23 101.46 16.57 5.56
60 108.06 109.09 106.98 104.09 104.42 103.23 98.64 98.10 96.76 94.09 94.18 93.52 100.93 12.60 5.66
90 108.69 108.19 105.53 103.49 104.16 102.32 98.21 97.11 95.75 93.41 94.86 92.53 100.35 6.20 5.71

8.3.2. CALCULATIONS& & EQUATION at Buffer stage .

AUCT- (10.7873) 1000 100
Acetylsalicylic acid (dissolved %) at Buffer stage = ------------------ x -------- x -------
6.2846 Dosage 1000
(Test or ref.)
Where;
AUCT : Peak area of tested tablet
Slope : 6.2846
Intercept : 10.7873
Dosage of Test : one tablet equivalent to 100 mg Acetylsalicylic acid
Dosage of Reference: one tablet equivalent to 100 mg Acetylsalicylic acid

Table 64: Summary of percent released of Acetylsalicylic acid from Test at Buffer Stage
Time
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 Mean SD RSD
(min)

15 50.81 52.78 48.09 52.22 51.29 47.69 54.02 46.61 54.60 51.24 52.38 53.20 51.24 10.34 4.98
20 66.56 63.14 65.45 66.99 52.30 64.44 65.51 67.79 75.92 68.88 76.32 73.24 67.21 14.46 9.53
30 72.31 83.73 84.96 78.45 76.09 83.05 60.09 83.95 81.36 69.93 85.48 78.67 78.17 15.20 9.69
45 80.82 82.44 84.57 78.86 80.80 84.13 86.91 89.95 86.37 84.81 88.81 83.40 84.32 11.36 3.95

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 60 80.13 84.59 84.21 78.19 80.48 82.47 88.41 90.30 87.74 86.28 88.95 84.46 84.68 7.77 4.52
90 80.28 82.70 82.15 80.55 80.15 81.31 91.24 89.70 89.47 87.71 88.22 85.46 84.91 4.24 4.91

Table 65: Summary of percent released of Acetylsalicylic acid from Reference at Buffer Stage
Time
R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12 Mean SD RSD
(min)

15 46.58 50.09 50.82 54.07 53.20 50.76 57.63 41.15 46.62 55.26 34.15 47.48 48.99 11.28 13.21
20 63.89 64.95 67.21 75.09 70.01 63.79 76.11 60.96 69.39 74.99 53.26 69.25 67.41 14.59 9.80
30 74.64 80.01 82.04 80.74 79.35 80.29 58.41 80.12 86.36 82.44 80.83 82.90 79.01 15.17 8.90
45 84.16 80.01 85.75 79.37 78.38 86.12 90.09 89.95 87.84 86.64 85.40 86.39 85.01 11.70 4.57
60 83.44 78.86 84.41 81.28 77.78 84.89 90.35 88.68 87.25 84.99 85.11 85.53 84.38 7.63 4.37
90 82.92 80.10 82.33 80.37 80.05 83.94 88.69 88.33 86.93 84.11 86.79 84.21 84.06 3.39 3.68

8.4. Similarity factor "f2" calculation for each pH

- Similarity factor (f2) = 50 × log (1 + 1/n∑ n (Rt – Tt ) 2 )- 0.5 ×100

- Similarity factor (f2) should be ≥ 50.

8.4.1. Similarity factor (f2) at pH 6.8

Similarity factor (f2)= 70.74

8.4.2. Similarity factor (f2) at Buffer stage .

Similarity factor (f2)= 92.05

8.5. Dissimilarity factor "f1" calculation for each pH

-Dissimilarity factor (f1) = { [ ∑ Rt-Tt ] [ ∑ n Rt ] }٠ 100

-Dissimilarity factor (f1) should be ≤15

8.5.1. Dissimilarity factor (f1) at pH 6.8

Dissimilarity factor (f1)= (90.5-88.1)/88.1=2.73

8.5.2. Dissimilarity factor (f1) at Buffer stage .

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 Dissimilarity factor (f1)= (75.0-74.8)/74.8=0.24

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8.6. Comparative dissolution profile for each pH

8.6.1 Dissolution Profile of Acetylsalicylic acid from Rivo 100 mg Enteric Coated
Tablets against Asprin® protect 100 mg at pH 6.8

Table 66: Dissolved (%) drug from generic and reference product at pH 6.8

Amount (%) of Acetylsalicylic acid released at pH 6.8

Time Generic
(min) Reference
(Rivo 100 mg Enteric
(Asprin® protect 100 mg )
Coated Tablets.)
15 60.14 54.51
20 83.58 76.91
30 94.89 94.67
45 100.48 101.46
60 101.54 100.93
90 102.62 100.35
Dissimilarity factor (F1) 2.73
Similarity factor (F2) 70.74

Figure 7: Dissolution Profile of Acetylsalicylic acid in dissolution medium at pH 6.8

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 8.6.2 Dissolution Profile of Acetylsalicylic acid from Rivo 100 mg Enteric Coated
Tablets against Asprin® protect 100 mg at Buffer stage

Table 67: Dissolved (%) drug from generic and reference product at buffer stage

Amount (%) of Acetylsalicylic acid released at buffer stage

Time Generic
(min) Reference
(Rivo 100 mg Enteric
(Asprin® protect 100 mg )
Coated Tablets.)
15 51.24 48.99
20 67.21 67.41
30 78.17 79.01
45 84.32 85.01
60 84.68 84.38
90 84.32 84.07
Dissimilarity factor (F1) 92.06
Similarity factor (F2) 0.24

Figure 8: Dissolution Profile of Acetylsalicylic acid in dissolution medium at buffer stage

9. Dissolution method validation

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9.1 Validation report for the most suitable medium (Buffer Stage)

9.1.1 Calibration curve (with regression equation)

Table 68: Calibration curve for Acetylsalicylic acid (25 - 200 µg/ml) at Buffer stage
Average Peak Area Response for Different Concentrations of Acetylsalicylic acid
(25 – 200 µg/ml)

Conc. Peak Peak Peak Average RSD

(µg/ml) Area 1 Area 2 Area 3 Peak Area
25 167.3 167.2 166.4 166.97 0.30
50 326.2 325.9 325.8 325.97 0.06
75 480.2 479.3 478.8 479.43 0.15
100 641.5 641.0 641.0 641.17 0.05
150 956.3 955.8 958.4 956.83 0.14
200 1267.1 1264.3 1264.0 1265.13 0.14

Figure 9: The calibration curve of Acetylsalicylic acid at Buffer stage

9.1.2 Linearity

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Results & Statistics
Regression line equation Y = Bx ± A
Intercept A = 10.7873
Slope B (variable) = 6.2846
R² (correction coefficient) =1.0000

Limit of Detection (LOD) = 3.3 x δ (standard deviation) / Slope = 0.420 µg/ml

Limit of Quantitation (LOQ) =10 x δ (standard deviation) / Slope = 1.270 µg/ml

Comment: Linear relationship was obtained between the recorded peak areas and their
corresponding concentrations. The correlation coefficient was found to be 1.0000.

9.1.3 Selectivity

Selectivity of Acetylsalicylic acid in the presence of its degradation products and other
additives:
The sample solution and the degraded sample solution were analyzed by HPLC to
determine that method could separate acetylsalicylic acid in presence of its degradation products
and other additives. The degraded sample exhibit different retention time with good resolution.

9.1.4 Accuracy

[Link] Accuracy
[Link].1 Definition
The accuracy of an analytical procedure expresses the closeness of agreement between the
value which is accepted either as a conventional true value or an accepted reference value and the
value found. This is sometimes termed trueness.

[Link].1 .2 Demonstration
Accuracy is usually demonstrated by adding known amounts of standard to the sample
matrix and determining the measured result using the analytical procedure. The recovery of
measured against actual amounts is then calculated. Usually a minimum of three determinations
at each of three concentrations across the intended range is recommended.

[Link].1.3 Data of Accuracy of Acetylsalicylic acid at buffer stage

Table 4: Acetylsalicylic acid Accuracy Data

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 Acetylsalicylic acid(µg/ml)
No.
Standard Total
Test Found Recovery (%)
Added (Theoretical)
1 78.53 40.00 118.53 118.33 99.83%
2 78.53 80.00 158.53 160.61 101.31%
3 78.53 120.00 198.53 197.90 99.68%
Mean Standard
100.27 % 0.90
Results & value: deviation:
Statistics Relative Standard
1.80%
Deviation
Comment: This study confirmed that the present method for determination of Acetylsalicylic
acid is specific and accurate at buffer stage.

9.1.5 Precision (interday and intraday)

[Link] Intermediate Precision of Acetylsalicylic acid

Data of Intermediate Precision of Acetylsalicylic acid
Table 70: Precision Data & Statistical Analysis of Acetylsalicylic acid at buffer stage
Individual value of
Test Individual value at
Acetylsalicylic acid
Parameters buffer stage
No. (µg/ml) at buffer stage
(1st day) (2nd day) (1st day) (2nd day)
1 99.91 100.30 Mean: (X) 100.046 99.180
2 99.83 100.58 Variance 0.5137 2.2687
3 99.64 100.75 Standard Deviation 0.717 1.506
4 101.46 97.77 Relative Standard 0.72% 1.52%
5 99.48 98.07 F-test (α =0.05)
4.417
6 99.95 97.61 F critical = 5.05

Comment: Six different samples were assayed in two different days, results obtained are
reported in Table (70).The results obtained were compared statistically (F-test) and showed that
no significant difference was obtained between results of two days .

[Link] Ruggedness

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 Data of Repeatability & Ruggedness Precision of Acetylsalicylic acid at buffer stage

Table 71: Acetylsalicylic acid Repeatability, Ruggedness & Statistical Analysis Precision Data

Individual value of
Test Individual value at
Acetylsalicylic acid
Parameters buffer stage
No. (µg/ml) at buffer stage
(1 Analyst)
st
(2nd Analyst) (1st Analyst) (2nd Analyst)
1 99.91 101.51 Mean: (X) 100.046 100.480
2 99.83 101.79 Variance 0.5137 1.3359
3 99.64 100.96 Standard Deviation 0.717 1.156
4 101.46 99.13 Relative Standard 0.72% 1.15%
5 99.48 100.36 F-test (α =0.05)
2.601
6 99.95 99.13 F critical = 5.05

Comment Six different samples were assayed by two different analysts; Results obtained are
reported in Tables (71) .The obtained data were subjected to statistical analysis (F-test) and
showed that the assays performed by the two analysts have the same degree of precision and
are equivalent.

9.1.6 Recovery

[Link] Spike – Recovery at (40%-120%-180%) of the Labelled Concentration:

Recovery is similar to accuracy but includes the extraction efficiency of an analytical
method. Recovery experiments should be performed by comparing the analytical results for
extracted samples at three concentrations (low, medium, and high) with unextracted standards
that represent 100% recovery. The data is summarized in the following table (72).

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 Table 72: Acetylsalicylic acid recovery at buffer stage
Acetylsalicylic acid(µg/ml)
No. Spiked Conc
(µg/ml) at Area Recovery (%)
buffer stage
265.8
1 40.0 259.8 102.18%
261.3
772.3
2 120.0 781.8 102.50%
786.9
1166.2
3 180.0 1164.0 102.25%
1172.8

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 CONCULSION

The study showed that the biowaiver for hard gelatin capsule dosage form containing
Acetylsalicylic acid is scientifically provided that the generic “Rivo 100 mg Enteric
Coated Tablets and innovator product “Asprin® protect 100 mg Gastroresistant
Tablets. dissolved with similarity of the dissolution profiles demonstrated at The most
Suitable media (Acid stage& Buffer Stage),pH 1.2, 4.5, 6.8) dissolved as described in the
BP procedure. When all of these conditions can be fulfilled, Pharmaceutical Equivalence
together with in vitro Similarity (IVS) will be suitable promising to indicate
Bioequivalence.

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