2.

1Drug profile

Drug profile of Aceclofenac:

Aceclofenac is a nonsteroidal anti-inflammatory drug (1) (NSAID) analog of diclofenac. It is

used for the relief of pain and inflammation (2) in rheumatoid arthritis (3), osteoarthritis
(4) and ankylosing spondylitis (5). Aceclofenac should not be given to people with porphyria
(6,7) or breast-feeding (8) mothers, and is not recommended for children. It should be
avoided near term in a pregnant woman because of the risk of having a premature closure of
ductus arteriosus (9) leading to fetal hydrops (10) in the neonate.
Structure of Aceclofenac [Fig. 2.01]:

Fig 2.01: Aceclofenac ’s chemical structure

2.1.2 Name of the IUPAC:

2-[2-[2-(2,6-dichloroanilino)phenyl]acetyl]oxyacetic acid

2.1.3Molecularformula: C16H13Cl2NO4

2.1.4 Molecular weight: 354.18 g·mol−1

2.1.5 Category: Aceclofenac is classified as a BCS Class II, poorly soluble and highly
permeable medication, and is used to treat pain. Prostaglandins (PG) are responsible for pain,
edoema, inflammation, and fever. Aceclofenac acts by blocking the enzyme cyclooxygenase
(COX).

2.1.6 Mechanism of action:11111111111111111111111111111111111111111111111111:

Aceclofenac reduces the generation of inflammatory mediators such as prostaglandin E2
(PGE2), IL-1, and TNF from the arachidonic acid (AA) route by inhibiting COX-2, a key
enzyme in this pathway. Diclofenac, which is converted from aceclofenac 6, is hypothesised
to have a role in the inhibition of IL-6. Reactive oxygen species generation is decreased when
inflammatory cytokines are suppressed. This drug has been found to reduce the formation of
nitrogen oxide in human joints by aceclofenac. aceclofenac also inhibits neutrophil adherence
to endothelium by reducing the expression of L-selectin (CD62L), a cell adhesion protein
produced on lymphocytes. Glycosaminoglycan synthesis in human osteoarthritic cartilage
may be facilitated by aceclofenac's ability to decrease IL-1 production and activity.

CHAPTER-2 Page 35
 By suppressing the IL-1-induced synthesis of promatrix metalloproteinases-1 and-3, 4'-
hydroxyaceclofenac provides chrondroprotection by inhibiting the release of proteoglycan
from chrondrocytes.

2.1.7 Side effects of Aceclofenac:

 Abdominal pain.
 Constipation.
 Diarrhoea.
 Nausea.
 Vomiting.
 Skin rash.
 Dizziness.
 Visual Disturbance.

2.1.8 Contraindications:
 Ischaemic heart disease.
 Peripheral arterial disease.
 Cerebrovascular disease.
 Congestive heart failure (New York Heart Association, NYHA, classification II-IV)

2.1.9 Absorption: Aceclofenac is promptly and fully absorbed as an unmodified medication

after oral administration. 1.25 to 3.00 hours after intake, peak plasma concentrations are
obtained in the blood. There are roughly 57 percent more concentrations of aceclofenac in
synovial fluid than there are in blood plasma.

2.1.10 Uses:

To alleviate discomfort, aceclofenac is prescribed. One of the most often prescribed

medications for rheumatoid arthritis, ankylosing spondylitis, and osteoarthritis. Aceclofenac
is a non-steroidal anti-inflammatory medication (NSAIDS).

2.2 Misoprostol
Drug profile of Misoprostol:

Misoprostol is a synthetic prostaglandin (11) medication used to prevent and treat stomach
ulcers, start labor, cause an abortion, and treat postpartum bleeding due to poor contraction of
the uterus Misoprostol is taken by mouth when used to prevent gastric ulcers in persons
taking NSAIDs. For abortions it is used by itself and with mifepristone or methotrexate
(12). By itself, effectiveness for abortion is between 66% and 90% (13,14). For labor
induction or abortion, it is taken by mouth, dissolved in the mouth, or placed in the vagina
(15). For postpartum bleeding it may also be used rectally (16). Common side effects
include diarrhea and abdominal pain. It is pregnancy category X meaning that it is known to
result in negative outcomes for the fetus if taken during pregnancy. In rare cases, uterine
rupture (17) may occur. It is a prostaglandin analogue (18) specifically, a
synthetic prostaglandin E1 (PGE1).

CHAPTER-2 Page 36
2.2.1 Structure of Misoprostol [Fig 2.02] :

Fig 2.02: Misoprostol chemical structure

2.2.2 Name of the IUPAC: methyl 7-[(1R,2R,3R)-3-hydroxy-2-[(E)-4-hydroxy-4-

methyloct-1-enyl]-5-oxocyclopentyl]heptanoate

2.2.3 Formula molecular: C22H38O5

2.2.4 Molecular weight: 382.541 g/mol-1

2.2.5 Category: Gastrointestinal Agents, Other; Prostaglandins, Endocrine include Cytotec in

their family of medications.

2.2.6 Mechanism of action: Synthetic prostaglandin E1 analogue misoprostol inhibits gastric

acid output by stimulating prostaglandin E1 receptors on stomach parietal cells. 3 Mucosal
bilayer thickening and increased mucus and bicarbonate secretion allow the mucosa to
produce more new cells. 3 By binding to uterine smooth muscle cells, misoprostol increases
contraction force and frequency while also degrading collagen and lowering cervical tone. 3

2.2.7 Side effects of Misoprostol :

 Diarrhea.
 Abdominal pain.
 Headache.
 Severe allergic reaction (anaphylaxis)
 Anemia.
 Abnormal heart beat.
 Chest pain.
 Gas (flatulence)

2.2.8 Contraindications:
Anticoagulant treatment, bleeding disorders, and ectopic or molar pregnancies are all
examples of contraindications for a misoprostol-induced abortion procedure.

2.2.9 Absorption:

CHAPTER-2 Page 37
Misoprostol is frequently undetectable in plasma because of its fast de-esterification prior to
or during absorption. The active metabolite of misoprostol, misoprostol acid, has a clearance
rate of 0.286 litres per kilogramme per minute.

2.2.10 Uses:

Preventing ulcers in patients who use aspirin and other arthritis or pain medications may be
done with the aid of misoprostol. In addition, it reduces the amount of stomach acid
produced.
2.2.11 Adult dose:
 100mcg
 200mcg
2.3 Literature resurvey

,
B. Jagadeesh Naidu K.E. Pravallika, Ravi Parimi, Validation of a new stability-
indicating RP-HPLC technique for simultaneous measurement of aceclofenac in bulk
and misoprostol in their combination dose form: Using reverse phase technology, we
developed and validated a technique for simultaneous determination of the bulk and
combination tablet formulation of Aceclofenac and Misoprostol, as well as stability tests for
both formulations. Aqueous 0.01M triethylamine buffer (pH 2.5) and acetonitrile: aqueous
0.01M triethylamine buffer (pH 2.5) were used as the mobile phase and the flow rate was set
at 1 ml/minute. The eluent was monitored by UV detector wavelength at 227 nm in high-
performance liquid chromatography (HPLC) with isocratic elution. There were 2.541 and
3.831 minutes of retention for Aceclofenac and Misoprostol, respectively; the LOQ was
0.250 and 0.255 nm; the correlation coefficient was 0.98 and 0.999; and the LOD was 0.125
and 0.127 nm. Aceclofenac and Misoprostol were intended to have a linearity range of 0.5-
7.52 g/ mL and 0.51-7.56 g/ mL, respectively. Relative standard deviation (percent RSD)
values of 0.21 and 0.28 for Aceclofenac and Misoprostol in accuracy studies indicated
percentage recovery rates of 100.1% to 100.8 percent and 100.0% to 100.4 percent.
According to ICH guidelines, the new approach was verified and confirmed to be optimum
and optimal for routine laboratory analysis.

Kanhaiyalal Patidar, Jigar Mehta, Vipul Patel , Nayan Kshatri and Niranjan Vyas, An
in vitro dissolving technique for misoprostol with HPLC analysis was developed and
validated: A reverse-phase liquid chromatographic technique will be used to create and verify
a dissolving test for misoprostol tablets comprising 200 g of misoprostol [1 percent in
HPMC]. It was found that using a 500-ml deaerated water dissolving medium and swirling it
at a speed of 50 revolutions per minute (rpm), together with sink conditions and dissolution
mediums of various densities, provided optimal circumstances for the stability of the
medication. Specificity, precision, linearity, and accuracy were all evaluated as part of the
method's validation in preparation for submission to international regulatory agencies for
approval. Over the course of the trial, more than 85% of the label quantity was released in the
medium. Using the dissolvability test devised, the dosage form of misoprostol may be tested
for quality control.
D.Vijaya Bharathi, B. Jagadeesh Kishore Kumar, Hotha Uday Patil, Indu Bhushan,
Method development and validation for the detection of misoprostol free acid in human
plasma by liquid chromatography–electrospray ionisation tandem mass spectrometry,
with application to a clinical pharmacokinetic investigation: For the determination of

CHAPTER-2 Page 38
misoprostol free acid in human plasma utilising misoprostol acid-d5 as an internal standard, a
highly sensitive, selective, and evaporation-free SPE extraction, ESI-LC–MS/MS approach
was established (IS). Using isocratic mobile phase on a reverse phase column and the
corresponding [MH] anions with a mass range of 367–249 m/z for misoprostol acid and 372–
249 m/z for IS, the analyte was separated and analysed by MS/MS in the multiple reaction
monitoring mode. Misoprostol acid and misoprostol acid-d5 (IS) were eluted after 3.6
minutes of running time. A quantitative limit of 2.5 pg/mL was established for the newly
proposed approach in human plasma. Misoprostol acid in human plasma exhibited a linear
response function (r > 0.998) for the concentration range of 2.5–1200 pg/mL. It was
determined that the FDA-accepted precision values for misoprostol acid were acceptable on
an intra- and inter-day basis. Benchtop, auto-sampler, and freeze/thaw cycles all found
misoprostol acid to be stable. An oral pharmacokinetic investigation in humans using the
newly designed test technique.
HuiyingSongGetuKahsay,FranEerdekens,YaxinTie,DannyHendriks,AnnVanSchepdaele

t al, Misoprostol-related compounds and diastereoisomers are being separated using

LC techniques that have been developed and validated: In addition to being used to treat
and prevent stomach ulcers, misoprostol may be utilised to induce labour during an abortion
owing to its labor-inducing properties. Many contaminants are present because of the drug's
instability at higher temperatures and moisture levels. Misoprostol occurs as a combination of
diastereoisomers (1:1). Reversed phase liquid chromatography (RPLC) and normal phase
liquid chromatography (NPLC) methods are given for the separation of the related
compounds and misoprostol diastereoisomers, respectively. An Ascentis Express C18 (150
mm x 4.6 mm, 5 m) column was used for the RPLC technique. At a flow rate of 1.5 mL/min,
the mobile phase was a gradient mixture of 28:69:3 v/v/v ACN–H2O–MeOH and 47:50:3
v/v/v ACN–H2O–MeOH. Detection was made at a wavelength of 200 nm. At 35 °C, an
XBridge bare silica column was used to perform the NPC technique. 1-propanol–heptane–
TFA (4:96:0.1% v/v/v) was injected into the mobile phase at 0.5 mL/min. At 205 nm, UV
detection was carried out. The two diastereoisomers (Rs > 2) may be separated in less than 20
minutes using this LC approach. The ICH criteria were followed in the validation of both
approaches. As a result, these novel LC techniques have been used effectively to monitor the
purity and diastereoisomers ratio of misoprostol bulk medication, tablets, and dispersion.
2.4 Experimental

2.4.1 Chemical and reagents:

Merck India Ltd. in Mumbai, India provided the acetonitrile, HPLC-grade formic acid, and
water needed for this experiment. Glenmark in Mumbai provided the APIs for the standards
for Aceclofenac and Misoprostol.

2.4.2 Instrumentation:

For this work, we employed a Waters Alliance liquid chromatography (model e-2695) that
was monitored by the empower 2.0 data management system and a light diode array detector
(model 2998).

2.4.3 Preparation of standard solution

Use 50 mg Aceclofenac and 50 mg Misoprostol as working standards, and put them in a 100
ml flask before diluting them with diluents to the desired concentration. Dilute the prepared
solution to a final volume of 50 ml by adding diluents to a further 5 ml.

CHAPTER-2 Page 39
Preparation of sample solution
Assemble a flask with 100 ml of diluents and 69.5 mg of Aceclofenac and Misoprostol
sample in it, then sonicate to dissolve it. A further 5 ml of the sample solution is diluted to 50
ml by adding diluents to the sample solution.

2.5 Method Development

Analytical method development:

Accuracy for Aceclofenac and Misoprostol analysis has been established using RP-HPLC in
the proposed research.

2.5.1 Method development parameters:

Selection of following parameters in method development is very important.

 Mode of chromatography
 Wavelength
 Column
 Mobile phase composition
 Solvent delivery system
 Flow rate
 Injection volume

2.5.1.1 Selection of mode of chromatography:

Selected mode of chromatography: Reversed phase chromatography

Basis of selection : polarity of the molecule

Reason for selection : as Aceclofenac and Misoprostol is polar molecule it

elutes at faster along with mobile phase

2.5.1.2 Detector wavelength selection:

The choice of a detector wavelength is critical to completing the analytical procedure. PDA
detector and wavelength are used to determine precise wavelength of the standard API, which
is manufactured and injected into the chromatographic system using PDA detector and
wavelength.

Selected wave length: 227 nm

Basis for selection: Maximum absorbance of analytes and impurities

Reason for selection: Aceclofenac and Misoprostol having maximum absorbance 227 nm.

2.5.1.3 Selection of column:

Column selected: Inertsil ODS (150x4.6mm, 3.5 µ)

CHAPTER-2 Page 40
Basis for selection: Based on the polarity, and chemical differences among analytics

Reason for selection:

Excellent physiochemical surface characteristics and compatibility with a wide variety of

organic solvents are only some of the advantages of these materials.

2.5.1.4 Selection of the mobile phase composition and of the buffer:

Peak symmetries and separation are heavily influenced by the buffer and its intensity. There
are a number of factors that must be taken into consideration when selecting the proper buffer
strength for a chromatographic injection load.

Mobile phase preparation:

Solution A: Acetonitrile

Solution B: 0.1percent formic acid

2.5.1.5 Selection of the rate of flow:

Even in reverse phase separation for the resolution of tiny molecules, flow rate is a crucial
element. However, in large-scale inverted phase chromatography, adding the sample solution
at a high flow rate has a significant impact on the analytical results. The dynamic binding
capacity of a sample might vary depending on the flow rate employed for sample loading.
When scaling up the purification process, the dynamic binding capacity must be estimated to
determine the optimal flow rate for loading. In this system, the flow rate is set to 1 ml/min
and is dependent on factors such as flow factor, retention duration, column composition,
separation impurity, and peak symmetrical symmetry.

2.5.1.6 Selection of injection volume:

For API estimate, a volume of 10 to 20 l is often advised. Extraction proved problematic, thus
the test concentration may be reduced and the injection volume increased to 50 l in the end.
As long as the specified column volume isn't overflowing, it's all ok. 10 l of Aceclofenac and
Misoprostol are injected in this approach.

Results and Discussion

2.5.1.7 Trials in optimization of chromatographic condition:

Trial-1 [Fig 2.03]

CHAPTER-2 Page 41
Buffer: Ammonium acetate pH-4 adjusted with OPA

Mobile phase: Acetonitrile and Buffer (80:20)

Column: Agilent eclipse XDB (250x 4.6mm, 5µ)

Rate of flow: 1ml/min

Volume of injection: 10µl

Period of run: 15 min

Wavelength: 200-400 nm

Observation: Only one peak observed

0.18 6.248

0.16

0.14

0.12

0.10
AU

0.08

0.06

0.04

0.02

0.00

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00
Minutes

Figure 2.03: chromatogram of trial-1

Trial-2 [Fig 2.04]

Buffer: Ammonium acetate pH-4 adjusted with OPA

Mobile phase: Acetonitrile and Buffer (70:30)

Column: Agilent eclipse XDB (250x 4.6mm, 5µ)

Rate of flow: 1ml/min

Volume of injection: 10µl

Period of run: 9 min

Wavelength: 227 nm

Observation: Merged peak is obtained

CHAPTER-2 Page 42
 2.725
3.00

3.122
2.00
AU

1.00

0.00

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

Minutes

Figure 2.04: Chromatogram of trial-2

Trial-3 [Fig 2.05]

Buffer: Ammonium acetate pH-4 adjusted with OPA

Mobile phase: Acetonitrile and Buffer (60:40)

Column: Agilent eclipse XDB (250x 4.6mm, 5µ)

Rate of flow: 1ml/min

Volume of injection: 10µl

Period of run: 15min

Wavelength: 227 nm

Observation: Base line is not sufficient

0.035
6.832

0.030
6.560

0.025

0.020
AU

0.015

0.010

0.005

0.000

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00
Minutes

Figure 2.05: Chromatogram of trial-3

Trial-4 [Fig 2.06]

Mobile phase: Acetonitrile and 0.1% OPA (35:65)

CHAPTER-2 Page 43
Column: Agilent eclipse XDB (250x 4.6mm, 5µ)

Rate of flow: 1ml/min

Volume of injection: 10µl

Period of run: 7min

Wavelength: 227 nm

Observation: Broad peaks are obtained

1.20

4.581
2.781
1.00

3.150
0.80
AU

0.60

0.40

0.20

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50
Minutes

Figure 2.06: Chromatogram of trial-4

Trial-5 [Fig 2.07]

Mobile phase: Acetonitrile: 0.1% OPA (70:30)

Column: Inertsil ODS (150x4.6mm, 3.5 µ)

Rate of flow: 1ml/min

Volume of injection: 10µl

Period of run: 7min

Wavelength: 227 nm

Observation: Resolution is not within the limit

0.040
2.340

3.560

0.030
AU

0.020

0.010

0.000

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

CHAPTER-2 Page 44
 Figure 2.07: Chromatogram of trial-5

Trial-6 [Fig 2.08]

Mobile phase: Acetonitrile: 0.1% OPA (50:50)

Column: Inertsil ODS (150x4.6mm, 3.5 µ)

Rate of flow: 1 ml/min

Volume of injection: 10µl

Period of run: 10 min

Wavelength: 227 nm

Observation: This method is suitable for validation

Figure 2.08: Chromatogram of trial-6

The chromatographic results of all trials were represented in the following table 2.01.

USP USP UUSP

Trial No. RT Area % Area
Resolution Tailing Plate count
1 6.248 16375076 100.00 1.06 91
2.725 684523 72.64 2.54 682
2
3.122 274563 24.37 4.28 3.42 1785
6.560 131075 50.57 1.19 8734
3
6.832 128116 49.43 1.29 1.15 44525
4 2.781 257415 18.74 1.08 1826

CHAPTER-2 Page 45
 3.180 481136 75.26 4.15 1.02 5263
4.581 321523 23.27 5.22 3.55 1863
2.340 130185 20.67 1.23 6318
5
3.560 499668 79.33 5.37 1.13 1806
3.163 3365478 1.15 0.96 3654
6
6.971 3469581 1.08 5.27 1.14 8547
Table 2.01: Chromatographic results of all trials

2.5.2 Optimized method [Table 2.02, Fig 2.09]

S.N
Parameter Chromatographic condition
O
1 Mobile phase Acetonitrile: 0.1% OPA (50:50)
2 Column Inertsil ODS (150x4.6mm, 3.5 µ)
3 Rate of flow 1ml/min
4 Column temperature Ambient temperature
5 Sample temperature Ambient temperature
6 Wavelength 227 nm
7 Volume of injection 10µl
8 Period of run 10 min
Aceclofenac Retention time-3.163
9 Retention time
Misoprostol retention time-6.971
Table 2.02: Optimized method chromatographic conditions

Figure 2.09: Chromatogram of standard

2.6 validation of method

It has been shown to be accurate, specific and precise in compliance with the ICH Q2 (R1)
requirements for device appropriateness, as well as robustness, LOD, LOQ and stability.

2.6.1 Specificity [Fig 2.10]

Specificity is the ability to test the analyte in the presence of other components, such as
contaminants or excitements, that may be presumed to be present in the sample solution and

CHAPTER-2 Page 46
the norm solution, without any interference. A blank sample was used as a control and spiked
with Aceclofenac and Misoprostol.

Figure 2.10: Chromatogram of blank

2.6.2 Linearity:

As a consequence of its capacity to deliver findings within a predetermined context, the

empirical technique is linear. It was determined that the peak area correlated with the
concentration of analytes in the sample: six standard solutions were used. The peak area was
displayed on the calibration curve and the regression equations were derived using the normal
solution concentration as a reference point. The least squares technique of least squares was
used to calculate the slope, intercept, and correlation coefficient.

Linearity stock solution preparation:

Use 50 mg Aceclofenac and 50 mg Misoprostol as working standards, and put them in a 100
ml flask before diluting them with diluents to the desired concentration.

10 percent solution preparation:

Dilution of the stock solution in a new 50 ml volumetric flask was carried out using the
diluents stated above.

25 percent solution preparation

One of the stock solutions was diluted in a 50 ml volumetric flask with diluents to the mark in
another 50 ml volumetric flask.

50 percent solution preparation

2.5 ml of the aforesaid stock solution was diluted with the diluents up to the mark in a
separate 50 ml volumetric flask.

CHAPTER-2 Page 47
100 percent solution preparation

Stock solution was diluted in a 50-ml volumetric flask with the diluents up to the mark in a
separate 50-ml volumetric flask.

125 percent solution preparation

6.25 ml of the above-mentioned stock solution was diluted with the diluents to the
appropriate concentration in a separate 50 ml volumetric flask.

150 percent solution preparation

7.5ml of the above-mentioned stock solution was diluted with the diluents to the appropriate
concentration in another 50 ml volumetric flask.

Procedure:

Using a chromatographic technique, measure the peak area for each degree. Plotting the peak
area against the concentration (on the X-axis) and then calculating the correlation coefficient
is what this method entails. Results of linearity is in table 2.03 and calibration plots were in
fig 2.11, 2.12.

Range:

To put it another way: The range of analytic approaches encompasses the gap between the
top and lower levels of analysis.

Acceptance criteria:

The correlation coefficient should not be less than 0.999

Analyte Range of Linearity calibration curve equation Correlation coefficient

Aceclofenac 5-75 µg/ml Y=68390.79x+14397.06 0.99998
Misoprostol 5-75 µg/ml Y=70351.52x+4707.45 0.9999
Table 2.03: Results of linearity

CHAPTER-2 Page 48
 6000000

Area Counts 5000000

f(x) = 66312.2219141021 x + 25421.9942509299
R² = 0.99954303265415
4000000
3000000
2000000
1000000
0
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00
Conc in ppm

Figure 2.11: Calibration plot of Aceclofenac

6000000
Area Counts

5000000
f(x) = 66312.2219141021 x + 25421.9942509299
R² = 0.99954303265415
4000000
3000000
2000000
1000000
0
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00

Conc in ppm

Figure 2.12: Calibration plot of Misoprostol

2.6.3 Accuracy:

50 percent solution preparation (with respect to the concentration of the target assay)

Recovery trials at three different concentration levels determined the accuracy (50 percent ,
100 percent and 150 percent ). APIs were produced at concentrations of 25, 50, and 75 g/ml.
The assay was carried out in accordance with the test procedure, in which the test solution
was injected into three preparations at each spike level. Aceclofenac and Misoprostol had
percentage recovery values of 99.5-100.6 and 99.4-100.4, respectively. Accuracy results were
in table 2.04.

CHAPTER-2 Page 49
Acceptance criteria:

The rate of recovery for each stage should be between 98-102 percent

Aceclofenac Misoprostol
% of target concentration
% Recovery % RSD % Recovery % RSD
50 99.6 1.19 99.4 0.39
100 100.6 0.64 100.2 0.47
150 99.5 0.35 100.4 0.58
Table 2.04: Accuracy results of Aceclofenac and Misoprostol

2.6.4 Precision:

In an analytical procedure, the rate at which repeated homogenous samplings provide similar
results is a measure of accuracy. Six injections of Aceclofenac (50ppm) and Misoprostol
(50ppm) were spiked to ensure the correctness of the injection method. Table 2.05 gives
results of system precision and the figures from 2.13-2.18 shows system precision
chromatograms.

Standard results
S.NO
Aceclofenac Misoprostol
1 3352468 3496582
2 3324175 3478512
3 3302659 3465001
4 3354628 3485796
5 3346291 3477519
6 3344570 3425631
Mean 3337465 3471507
Std dev 20179.790 24759.163
% RSD 0.60 0.71
Table 2.05: Results of system precision

Figure 2.13: Chromatogram of system precision-1

CHAPTER-2 Page 50
 Figure 2.14: Chromatogram of system precision-2

Figure 2.15: Chromatogram of system precision-3

Figure 2.16: Chromatogram of system precision-4

CHAPTER-2 Page 51
 Figure 2.17: Chromatogram of system precision-5

Figure 2.18: Chromatogram of system precision-6

Method precision [Table 2.06 and Fig 2.19]

Aceclofenac Misoprostol
S.NO
area area
1 3365104 3485796
2 3385676 3475162
3 3395864 3455216
4 3374856 3485126
5 3320156 3421365
6 3395694 3425163
Mean 3372892 3457971
Std dev 28483.927 29089.893
% RSD 0.84 0.84
Table 2.06: Results of method precision

Acceptance criteria: The RSD percent for the area six standard injection results should be
more than 2 %.

CHAPTER-2 Page 52
 Figure 2.19: Chromatogram of method precision

Acceptance criteria: The RSD percentage for the six normal injection results should not be
more than 2%.

2.6.5 Limit of detection (LOD) and limit of quantification (LOQ):

Using quality formulae, the limit of detection and quantification (LOQ) may be calculated as
the concentration below which an analyte can be reliably identified. Aceclofenac and
Misoprostol had LOD values of 0.06 g/ml and 0.06 g/ml, respectively, and s/n values of 5, 5.
As for Aceclofenac and Misoprostol, the LOQ was 0.208 g/mL for both drugs, and the s/n
was 26 for both.

2.6.6 Robustness:

The tactic's robustness was shown to attract RSD at a rate of just 2%. The optimum technique
parameters, such as flow (0.2 ml/min) and organic content in mobile phase (10 percent), were
wiped away by little adjustments. Table 2.07 gives results of robustness and the figures from
2.20-2.23 shows robustness chromatograms.

Table 2.07: Robustness study of Aceclofenac and Misoprostol

Flow Plus Flow Minus Org Plus Org Minus

Drug name (1.2 ml/min) (0.8 ml/min) (55:45) (45:55)
% RSD
Aceclofenac 0.35 0.19 0.69 0.54
Misoprostol 0.27 0.62 0.42 1.36
Table 2.07: Robustness study of Aceclofenac and Misoprostol

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 Figure 2.20: Less flow rate chromatogram (0.8ml/min)

Figure 2.21: More flow rate chromatogram (1.2ml/min)

Figure 2.22: Less organic chromatogram (45:55)

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 Figure 2.23: More organic chromatogram (55:45)

2.6.7 Forced degradation studies:

Studying 1 N concentrations of forced degradation settings that included acidic and basic
peroxides as well as hydrolysis and reduction, as well as thermal stress was done.

Stock solution preparation:

After correctly weighing and transferring the 50 mg of Aceclofenac and the 50 mg of

Misoprostol to the 100 ml volumetric flask, it is necessary to add the 70 ml of diluents and
sonicate for 30 minutes to completely dissolve the diluents.

Acid degradation:

For 15 minutes, add 1 ml of 1N Hcl to 1 ml of the sample stock solution in a volumetric flask
with a capacity of 10 ml. Add 1ml of 1N NaOH after 15 minutes and dilute to the desired
strength.

Alkali degradation:

For 15 minutes, 1 ml of sample stock solution is transferred to a 10 ml volumetric flask and 1

millilitre of 1N NaOH is added. After 15 minutes, add 1 ml of 1N Hcl and diluents to get the
solution to the desired volume.

Peroxide degradation:

Transferring 1ml of the sample stock solution to a 10ml volumetric flask, a 30 percent
hydrogen peroxide solution was added, and the volume was brought up to capacity using
various diluents.

Reduction degradation:

It was transferred to a 10-ml volumetric flask, where it was diluted with 30 percent sodium bi
sulphate solution and brought up to the diluents mark.

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Thermal degradation:

In the oven for six hours, the sample solution was kept at 105oC. HPLC was used to purify
the final product.

Hydrolysis degradation:

Sample stock was transferred to a volumetric flask of 10ml and diluted with 1ml of water to
get the desired amount of water dilution.

Table 2.08 gives results of forced degradation and the figures from 2.24-2.30 shows forced
degradation chromatograms.

Degradation Aceclofenac (% degradation) Misoprostol (% degradation)

Control 0.1 0.2
Acid deg 17.1 16.1
Alkali deg 16.4 15.9
Peroxide deg 14.6 13.5
Reduction deg 12.7 12.7
Thermal deg 13.2 10.8
Hydrolysis deg 11.8 11.9
Table 2.08: Forced degradation data of Aceclofenac and Misoprostol

Figure 2.24: Chromatogram of acid degradation

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 Figure 2.25: Chromatogram of alkali degradation

Figure 2.26: Chromatogram of peroxide degradation

Figure 2.27: Chromatogram of reduction degradation

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 Figure 2.28: Chromatogram of Hydrolysis degradation

Figure 2.29: Chromatogram of Thermal degradation

Figure 2.30: Chromatogram of control degradation

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Stability:

The stability of ordinary and sample solutions was examined from the beginning to the end of
a 24-hour storage period at room temperature by monitoring the stability methodologies.
They were given at various intervals, and the assay percentage variation between the original
and 24 hour injections was just around 2%. Aceclofenac and Misoprostol are not affected by
storage conditions. Table 2.09 gives results of stability and the figures from 2.31-2.35 shows
stability chromatograms.

Time intervals Aceclofenac (% Difference) Misoprostol % Difference

Initial 0.00 0.00
6 Hrs -0.9 -0.7
12 Hrs -1.1 -0.9
18 Hrs -1.7 -1.5
24 Hrs -2.8 -2.2
Table 2.09: Stability data of Aceclofenac and Misoprostol

Figure 2.31: Chromatogram of stability initial RT

Figure 2.32: Chromatogram of stability 6 Hrs RT

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 Figure 2.33: Chromatogram of stability 12 Hrs RT

Figure 2.34: Chromatogram of stability 18 Hrs RT

Figure 2.35: Chromatogram of stability 24 Hrs RT

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2.6.9 Conclusion:

In this procedure, Aceclofenac and Misoprostol are quantified according to ICH criteria in
bulk and pharmaceutical formulations. It was discovered that the newly developed method
was exact, accurate, linear, and dependable. The convenience and lower cost of the chemicals
used make this method more cost effective. To achieve reliable chemical quantification, the
recommended HPLC settings provide appropriate resolution. According to the findings of the
tests, the precision and repeatability data are good. The chromatographic technology that was
developed was extensively utilised in regular drug testing.

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