Method development and validation of Sodium Cromoglycate

PRESENTED BY :
Miss. Bhosale S. B.
M. Pharm. 2nd year
Dept. of Pharmaceutical Quality
Assurance
CO -GUIDED BY :
Miss. Wale R.R.
M. Pharm.
Dept. of Pharmaceutical
Chemistry.
GUIDED BY :
Dr. Gholve S. B.
M. Pharm., Ph.D.
Dept. of Pharmaceutical Quality
Assurance
CHANNABASWESHWAR PHARMACY COLLEGE (DEGREE), LATUR
for the fulfillment of award of degree of
MASTER OF PHARMACY
in

1. INTRODUCTION
2. LITERATURE OF REVIEW
3. AIM & OBJECTIVE
4. NEED OF STUDY
5. PLAN OF WORK
6. MATERIALS AND METHODS
7. OBSERVATION AND RESULTS
8. SUMMARY
9. CONCLUSION
10. REFERENCE
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Analytical method development is the process of selection of accurate assay method for the determination
of composition in a formulation.
Methods are developed to support drug testing against speciﬁcations during manufacturing and quality
release operations, as well as during long-term stability studies.
• Purpose of analytical method development ….
When there is no official drug or drug combination available in Pharmacopoeias.
The existing analytical procedures may need costly reagents and solvent.
When, there is no analytical method for the formulation of the drug due to the
interference caused by the formulation excipients.
The improvement of the analytical method development and analytical instrument
have reduce the time and cost of analysis and enhanced precision and accuracy.
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CHANNABASWESHWAR PHARMACY COLLEGE (DEGREE), LATUR 4
 Validation guidelines
1. ICH-Q2A “ Text on Validation of Analytical Procedure”: (1994)
2. ICH-Q2B “ Validation of Analytical Procedure : Methodology”:
(1995)
3. CDER “ Reviewer Guidelines: Validation of Chromatographic
Method” (1994)
4. CDER “Submitting Sample and Analytical Data for Method
Validation” (1987)
5. CDER Draft “ Analytical Procedure and Method Validation” (2000)
6. USP<1225>Validation of Compendial Methods (current revision)
7. ICH-Q1A “Stability study for drug substance and drug products”
8. ICH-Q1B “Photostability study”
 Validation parameters
1. Specificity
2. Range
3. Linearity
4. Limit of detection (LOD)
5. Trueness
6. Limit of quantitation (LOQ)
7. Precision
8. Robustness
9. Accuracy
10. Ruggedness
VALIDATION
Is the process of establishing documentary evidence demonstrating that a procedure, process, or activity
carried out in testing and then production maintain the desired level of compliance at all stages.
or
The act of officially or legally certifying or approving something. or validity of something. or to attest to
the truth.

SODIUM CROMOGLYCATE
Cromolyn sodium first discovered by Roger E. C. Altounyan in the early 1960s derived from a
healing herb called Ammi visnaga. Sodium cromoglycate used for the treatment of asthma and
other allergic disorders in both adults and children also.
Mechanism of Action : Sodium cromoglycate is an mast cell (present in eyes, nose, air passages,
along the digestive tract etc.) stabilizing agent. It works on certain cells in the body (mast cells) to
prevent them from releasing substances i.e. Histamine, leucotrines etc. that cause allergic
symptoms.
Side effects : Skin rash, Joint pain, Dizziness, Headache, Mouth and throat irritation, Unpleasant
taste, Blurred vision, Stinging or burning sensation of eyes.
Route of administration : Nesal spray, Nebulizer solution, Inhalers, Eye drops, Oral forms.
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Sr. No. Authors Description
1 Muhammad
F, et.al.
(2022)
This activity outlines the evaluation and treatment of asthma and explains the role of the inter
professional team in managing patients with this condition.
2 Alberto Papi
et.al. (2020)
Asthma is a common chronic disease characterized by episodic or persistent respiratory
symptoms and airflow limitation. The benefits of currently available treatments and the
possible strategies to overcome the barriers that limit the achievement of asthma control in
real-life conditions and how these led to the GINA 2019 guidelines for asthma treatment and
prevention will also be discussed.
3 Jaclyn Quirt
et.al.(2018)
Asthma is the most common respiratory disorder in Canada.
This article provides a review of current literature and guidelines for the appropriate
diagnosis and management of asthma in adults and children.
4 Christopher
Brightling,
et.al. (2017)
Asthma is a consequence of complex gene–environment interactions, with heterogeneity in
clinical presentation and the type and intensity of airway inflammation and remodeling. The
goal of asthma treatment is to achieve good asthma control- i.e. to minimize symptom
burden and risk of exacerbations.
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6

Sr. No. Authors Methodology Description
1 Maha A. Hegazy et.
al. (2018)
HPTLC and
HPLC
SP : C8 ( Rp 250×4mm, 5um)
Mp : ACN:Methanol:0.05M pot. dihydrogen phosphate =
20:30:50v/v/v
Flow rate : 1.2ml/min
Inj. Vol. : 20μL λ Max : 240nm
2 H. Elmansi et.al.
(2017)
IJPSR
Isocratic HPLC SP : C-18 (100mm×2.1mm, 1.7um)
MP : Methanol : OPA: ACN = 50:15:35v/v/v
Flow rate : 0.25ml/min, Inj. Vol. : 20μL . λ Max : 326nm
3 Magdy N et.al. (2016)
Analytical Chemistry
an Indian Journal
HPLC SP : C-18 (250×4.6mm, 5um)
MP: ACN : Water = 70:30
Flow rate : 1ml/min
Inj. Vol. : 10μl. λ Max : 235nm, RT : 11min
4 H. Elmansi (2016)
JCS
HPLC SP : C-18 (250mm×4.6mm, 5um)
MP: Methanol: 0.1M Phosphate buffer = 60:40v/v
Flow rate: 1ml/mil
Inj. Vol. : 20μL, λ Max : 220nm
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Sr. No. Authors Methodology Description
5 Maha A. Hegazy
(2015)
Journal of Advance in
Chemistry
Simultaneous
chromatography
SP: C-18(4.6×150mm, 5um)
MP : ACN : Methanol = 2:1v/v
Flow rate : 1ml/min, Inj. Vol. : 20μL, λ Max : 220nm
6 Paparaju. Sowjanya
et.al.(2013)
IJPRD
RP-HPLC SP : C-18 (250×4.6mm, 5um)
MP : ACN : pot.dihydrogen orthophosphate = 25:75
Flow rate : 1ml/min
Inj.Vol.: 20ul
λ Max : 240nm, RT : 5.1min
7 Sonia T. Hassib et.al
(2011)
JCPRCS
Stability
indicating RP-
HPLC
SP : C-18(250×4.6mm, 5um)
MP : Methanol : Water = 75:25
Flow rate : 1.2ml/min
Inj. Vol. : 100ul
λ Max : 254nm
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AIM
Spectroscopic & chromatographic method development and validation of sodium cromoglycate in
bulk and pharmaceutical dosage form.
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OBJECTIVES
• To develop a new simple, accurate, precise, rapid spectroscopic & chromatographic method for
sodium cromoglycate.
• To validate the developed method as per ICH guidelines.
• To minimize following parameters ; Cost, Time, Chemicals and Compatibility for the ICH
guidelines.
• To study forced degradation charecteristics of sodium cromoglicate by UV-visible
spectroscopy.

• There are numerous marketed products which are needed to analyze for the potency of
SCG in the product.
• To develop new economically cheap, simple, accurate and potent spectroscopic and
chromatographic method.
• To study the force degradation characteristics of SCG.
• To confirm the intended purpose of SCG through validation as per ICH guidelines.
• To identify, separate, and quantify the chemical components of medicinal products.
• To decrease the chemical quantity, time, cost of the solvents, and compatibility with ICH -
Q2 (R1) guidelines.
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Literature survey
Selection and procurement of drug
according to literature survey.
Whole drug profile
Selection of wavelength by critical examination of
UV absorbance spectra of the drug Selection of mobile phase
Method selection for chromatographic
analysis and working standards
concentration range
Validation of developed
method following ICH-
guidelines.
Statistical analysis and
calculation
Compilation of
results and
interpretation.
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Fig. 5.1. flow chart of plan of work CHANNABASWESHWAR PHARMACY COLLEGE (DEGREE), LATUR

DRUG PROFILE
Working standard /drug sample purchased from Rakshit pharmaceutical private
limited, Mumbai.
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O
O
O O
OH
O
O
O
O
O
O
Na
Na
IUPAC NAME : Disodium5-(3-(2-carboxylato-4-oxocromen-5-yl) –oxy-2-
hydroxypropoxy-) -4-oxocromen-2-carboxylate.
Synonyms : cromolyn, cromoglycate, cromoglicate, sodium
cromoglicate or cromolyn sodium.
Table 6.1. Drug profile of sodium cromoglycate

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Sr. No. Properties Description
3 Molecular formula C23H14O11Na2
4 Molecular weight 512.33 gm / mol
5 Melting point 260°C – 262°C
6 Log P 1.83
7 pKa 1.76
8 Bioavailability 1% Orally, 6-7% Inhalation spray.
9 Half Life 1.30hrs
10 Category Anti-asthmatic, Anti-allergic.
11 Mode of action Block the calcium channel and inhibit histamine
and leukotriene (SRS-A) release from mast
cell’s.
12 Route of
administration
Ophthalmic ( eye, nesal ), Oral.

CHEMICALS AND INSTRUMENTS
Sr. No. Chemicals Grade
1 Water Spectroscopic and HPLC grade
2 Acetonitrile HPLC grade
3 0.1% perchloric acid HPLC grade
Sr. No. Instruments Models
1 Wt. Balance Phoenix ISO 9001 Company
2 Sonicator Pci Analytical Sonicator
3 FT-IR PerkinElmer UATR Two
4 UV spectrophotometer Shimadzu UV -1800
5 HPLC Agilent 1220 infinity LCLC Shimadzu
6 pH meter BVK- Enterprises
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Table 6.2. Instruments & Models
Table 6.3. Chemicals & Grade

 DRUG AUTHENTIFICATION
1. Melting point : 261 - 263°C
2. Solubility : Freely soluble acetonitrile, water,
slightly soluble in methanol, then ethanol , insoluble
in ether.
3. FT - IR Spectroscopy : Identity of drug was
confirmed by comparing FT-IR spectra with sodium
cromoglycate (SCG) standard for presence of
functional groups.
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O
O
O O
OH
O
O
O
O
O
O
Na
Na
Fig. 6.1. Structure of SCG

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Functional groups
names
Standard
peak range
(cm-1)
Observed
peak (cm-1)
Aromatic CH3 group 3250-3100 3104.36
R-C=O aldehyde group 2970-2800 2841.56
C=C aromatic alkenes 1740-1590 1697.49
C-C aromatic stretching 1630-1400 1552.18
CH3 aliphatic group 1480-1350 1442.61
O -H Stretching 1450-1300 1317.86
C -O- Strong 1320-1190 1285.67
C - O -O- carboxylic
group
990-900 926.05
Administrator 845
Name
Sample 845 By Administrator Date Friday, March 25 2022
Description
4000 450
3500 3000 2500 2000 1500 1000 500
102
65
70
75
80
85
90
95
100
cm-1
%T
496.90cm-1, 65.84%T
611.52cm-1, 67.53%T
743.85cm-1, 70.07%T
1 6 5 4 . 8 9 c m - 1 , 7 1 . 8 6 % T
763.57cm-1, 72.28%T
1552.18cm-1, 74.57%T
1 4 4 2 . 6 1 c m - 1 , 7 5 . 7 8 % T
1416.10cm-1, 76.02%T
979.83cm-1, 76.44%T
813.68cm-1, 77.66%T
790.83cm-1, 78.12%T
1197.07cm-1, 79.37%T
926.29cm-1, 80.62%T
1227.99cm-1, 80.84%T
1242.86cm-1, 80.85%T
553.03cm-1, 80.85%T
953.82cm-1, 81.86%T
1285.67cm-1, 82.70%T
1317.86cm-1, 83.53%T
1697.49cm-1, 83.97%T
3 1 0 4 . 3 6 c m - 1 , 8 5 . 1 2 % T
1055.70cm-1, 86.36%T
1091.67cm-1, 87.52%T
2841.56cm-1, 89.70%T
Fig. 6.2. FT-IR spectra of SCG
Table. 6.4. Functional group ranges for SCG

SAMPLE PREPARATION PROCEDURE FOR UV
• Preparation of Standard stock solution :
10 mg drug + 100ml volumetric flask, make up the volume with MP ( ACN: Water = 80 : 20 ) i.e. 100µg /ml final
solution prepared. From above solution pipette out 10 ml and make up to 100 ml with MP to form 10µg/ml final
solution.
• Preparation of Working sample solution :
From above Standard stock solution pipette out 0.2ml, 0.4ml, 0.6ml, 0.8ml & make up the volume up to 10ml to
form 2µg/ml, 4µg/ml, 6µg/ml, 8µg/ml &10µg/ml respectively.
• Preparation of solution from marketed formulation (ASSAY) :
Label claim: Each ml of Eye drop contains 40 mg Sodium Cromoglycate ( Cromal forte 2% NaCr2 )
i.e. 0.125 ml contains 5 mg sodium cromoglycate so, 0.125 ml of eye drop was transferred in 10 ml volumetric
flask and mixed with 10 ml diluents. (Conc. = 500 µg/ml). Pipette out 1.0 ml of above solution in 10 ml
volumetric flask. Add 5 ml diluents and vortex, make up the volume with diluents. (Conc. = 50 µg/ml)
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SAMPLE PREPARATION PROCEDURE FOR RP-HPLC
• Preparation of Standard stock solution :
Initially Prepare a Standard Stock Solution (SSS-I) of Sodium cromoglycate by adding 5mg in 10 ml volumetric flask
& add 5 ml diluent and mix and sonicate for 5 minutes. Make up the volume to 10 ml with diluent. (Conc. = 500
µg/ml)
Pipette out 1.0 ml of SSS-I in 10 ml volumetric flask. Add 5 ml diluent and vortex; make up the volume with diluent.
(Conc. = 50 µg/ml).
• Preparation of Working solution :
From above standard solution pipette out 0.8ml, 0.9ml, 1ml, 1.1ml &1.2ml make up the volume with diluent up to
10ml to form 40µg/ml, 45µg/ml, 50µg/ml, 55µg/ml & 60µg/ml respectively.
• Preparation of 0.1% per chloric acid :
In a 1000 ml measuring cylinder, take 700 ml of HPLC grade Water, add 1 ml of perchloric acid and mix well, make
up to the mark with HPLC grade water. Filter twice using 0.45µ nylon filter membrane and sonicate to degas for
15min.
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 FOR BULK DRUG
• Selection of wavelength :
Standard stock solution of sodium cromolyn 10 µg / ml was prepared by using Acetonitrile :
Water (80:20) ratio. The maximum absorbance was observed at 272nm.
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Fig. 7.1. UV-visible spectra of SCG
7. OBSERVATIONS AND RESULTS

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 SYSTEM SUITABILITY
Single sample was prepared as described and 6 injections were made from same
sample and checked for system suitability, obtained results are ˂ 2 % RSD.
Sr. No
Conc. (µg/ml)
Absorbance
2
1 0.445
2 0.452
3 0.45
4 0.453
5 0.448
6 0.45
Average 0.44966666
SD 0.00287518
%RSD 0.63940277
Table 7.1. System suitability data for SCG

 Linearity study from the working standard at different conc. 2, 4, 6, 8, 10 µg/ ml of drug
solution was carried out. Absorbance was measured at 272 nm and results are recorded in
table 7.2.
y = 0.174x + 0.112
R² = 0.999
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0 5 10 15
Series1
Linear (Series1)
Sr. No. Conc.
(µg/ml)
Absorbance
1 2 0.45
2 4 0.816
3 6 1.18
4 8 1.492
5 10 1.856
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Table 7.2. Linearity data for SCG
Fig. 7.2. Linearity curve for SCG
Conc. µg/ml
Absorbance

 Inter-day precision :
Sr.no.
Conc.
(µg/ml)
Absorbance
2 6 10
1 0.45 1.18 1.856
2 0.454 1.2 1.81
3 0.465 1.84 1.805
4 0.459 1.18 1.83
5 0.46 1.23 1.85
6 0.457 1.19 1.84
Avg. 0.4575 1.194 1.831833333
SD 0.004764452 0.019183326 0.020884604
%RSD 1.041410208 1.606643726 1.140092999
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Table 7.3. Inter-day data for SCG
nm.
200.0 250.0 300.0 350.0 400.0
Abs.
0.540
0.400
0.200
0.000
-0.051
1
2
3
4
5
6
7
8
Fig. 7.3. Inter-day spectra of SCG

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nm.
200.0 250.0 300.0 350.0 400.0
Abs.
0.999
0.500
0.000
-0.094
1
2
3
4
5
6
7
8
9
10
11
Sr. No./
Conc.(µg/ml)
Absorbance
2 6 10
1 0.445 1.18 1.856
2 0.452 1.226 1.769
3 0.45 1.192 1.79
4 0.453 1.23 1.800
5 0.448 1.22 1.82
6 0.45 1.18 1.84
Avg. 0.44966666 1.20466666 1.8125
SD 0.00287518 0.02327803 0.032433008
% RSD 0.63940277 1.93232122 1.789407331
Table 7.4. Intra-day data for SCG
• Intra-day precision
Fig. 7.4. Intra –day spectra of SCG

 ROBUSTNESS
Sr. no / Conc.
(µg/ml)
Absorbance
2 6 10
1 0.41 1.15 1.799
2 0.41 1.13 1.84
3 0.42 1.16 1.85
4 0.41 1.17 1.847
5 0.40 1.12 1.841
Average 0.41 1.146 1.8354
SD 0.0070710 0.02073644 0.0207677
%RS D 1.7246506 1.80946259 1.1315115
• 1. Change in wavelength :
Sr. no /conc.
( µg/ml)
Absorbance
2 6 10
1 0.40 1.175 1.846
2 0.41 1.143 1.842
3 0.40 1.177 1.850
4 0.41 1.179 1.831
5 0.41 1.178 1.853
Average 0.406 1.1704 1.844
SD 0.0054772 0.0154023 0.00856154
%RS D 1.3490703 1.315966 0.46419116
Wavelength = 270 nm Wavelength = 274 nm
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Table 7.5. Robustness data of SCG at 270nm Table 7.6. Robustness data of SCG at 274nm

 LOD & LOQ
Sr. no. Parameters Data
1 λ Max 272nm
2 Linearity 2 - 10µg / ml
3 Regression
equation
Y =0.1744x+ 0.112
4 Correlation
coefficient (R2 )
R2 = 0.9998
5 Slop 0.1744
6 Intercept 0.1124
7 LOD 0.05440
8 LOQ 0.1648
1. LOD = 3.3 σ/S
2. LOQ = 10 σ/S
Where,
σ = standard deviation , (0.002875)
S= slope of calibration curve
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Table 7.7. LOD & LOQ data for SCG

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 ACCURACY
Accuracy
Conc. (µg/ml)
% Recovery
(AVG)
Statistical Analysis
Mean % RSD
80% 98.83 0.283667 1.876927
100% 101.16 0.379667 0.941611
120% 98.77 0.419333 1.31672
Table 7.8. Accuracy data for SCG

Sr. no. Stress condition Degradation Remark
1 Photolytic 12.40% Stable
2 Thermal 19% Stable
3 0.01N HCL 75.35% Unstable
4 0.01N NaoH 77.26% Unstable
5 H2O2
10% 21.43% Unstable
20% 23.60% Unstable
30% 26.53% Unstable
 FORCED DEGREEDATION STUDY
Force degradation study was carried out in different conditions, i.e. Acidic, Basic, Thermal,
Photolytic etc and the results were obtained. In all conditions except thermal and photolytic drug
was unstable.
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Table 7.9. Degradation data for SCG
nm.
200.0 250.0 300.0 350.0 400.0
Abs.
0.876
0.800
0.600
0.400
0.200
0.000
-0.083
1
2
3
4
5
6
7
8
Fig. 7.5. Thermal spectra of SCG

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nm.
200.0 250.0 300.0 350.0 400.0
Abs.
4.170
4.000
3.000
2.000
1.000
0.000
-0.313
1
2
3
4
5
6
7
8
9
10
11
12
nm.
200.0 250.0 300.0 350.0 400.0
Abs.
4.259
4.000
3.000
2.000
1.000
0.000
-0.381
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Fig. 7.6. Acid degradation of SCG Fig. 7.7. Base degradation of SCG
CHANNABASWESHWAR PHARMACY COLLEGE( DEGREE), LATUR

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For marketed formulation : (Cromal forte 4%)
Label claim: Each ml of eye drop contains 40 mg sodium cromoglycate.
i.e. 0.125ml contains 5 mg sodium cromoglycate. So, 0.125 of eye drop was
transferred in 10 ml volumetric flask and mixed with 10 ml diluent. (Conc. = 500
µg/ml).
Formula:
• % Assay = Sample area / Standard area ×100
• % Assay = 0.690 / 0.680 ×100
• % Assay = 101.47 %.
 ASSAY

RP-HPLC RESULTS
S. P. M. P. Diluent λ Max RT TP Asym.
Agilent Zorbax SB-Aq
(250 x 4.6mm, 5µ)
0.1% Perchloric acid:
Acetonitrile -50:50
50% 0.1% PCA : 50% ACN 250 2.92 554 0.73
(250 x 4.6mm, 5µ)
0.1% Perchloric acid :
Acetonitrile - 60:40
50% 0.1% PCA : 50% ACN 250 2.95 1576 0.83
(250 x 4.6mm, 5µ)
50% 0.1% PCA : 50% ACN 250 3.27 3469 0.77
(250 x 4.6mm, 5µ)
50% 0.1% PCA : 50% ACN 272 12.43 8992 1.10
(250 x 4.6mm, 5µ)
50% 0.1% PCA : 50% ACN 272 4.59 8876 1.11
Method development :
Depending on the wavelength, retention time, theoretical plate and asymmetry factor fourth no. of
method was optimized because of higher TP, lower retention time, less than 2 asymmetry factor.
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Table 7.10. Chromatographic Trials for Obtaining Optimized Methods

1
2
Fig. 7.8. Chromatographic Trails for SCG
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CHANNABASWESHWAR PHARMACY COLLEGE (DEGREE), LATUR 32
3 4

5
Fig. 7. 9. Optimized Chromatogram for SCG
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Sr. No. Parameters Conditions
1 Column Oven Temp. 30°C
2 Flow Rate 1 ml/min
3 Run Time 10 min
4 Inj. Volume 10 µL
5 Mobile Phase 0.1% perchloric acid : Acetonitrile,
80:20v/v
6 Diluent Acetonitrile: 0.1% perchloric acid,
50: 50v/v
7 Wavelength 272 nm
8 Column Agilent Zorbax SB-Aq (250 x 4.6
mm, 5µ)
 CHROMATOGRAPHIC CONDITIONS
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Table 7.11. chromatographic conditions for SCG

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 SELECTION OF WAVELENGTH
• From standard stock solution 10ug/ml sample was injected in HPLC system with PDA
detector. The observed maximum area of drug was at 272nm.
Fig. 7.10. Chromatogram of blank solution of SCG Fig. 7. 11. Chromatogram of standard solution of SCG

 LINEARITY
Linearity was determined for SCG in the range of 40 – 60ug/ml concentration. Linearity parameters
are observed and calculated as Slop, Range, & Correlation coefficient which pass the limits
according to ICH guidelines.
Sr. No Conc. (µg/ml) Area
1 40 2808897
2 45 3186270
3 50 3512722
4 55 3848126
5 60 4223381
y = 69816x + 25055
R² = 0.999
2500000
2700000
2900000
3100000
3300000
3500000
3700000
3900000
4100000
4300000
4500000
30 40 50 60 70
Area
Concentration (ug/ml)
Linearity
Fig.7.12. Linearity curve for SCG
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Table 7.12. Linearity data for SCG

Parameters Observation
Range 40-60µg/ml
Regression equation 69816.48x+ 25055.2
Correlation coefficient (r2) 0.9993
Slope 69816.48x
Intercept 25055.2
LOD 2.52
LOQ 7.64
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Table 7.13. LOD, LOQ data for SCG
 LIMIT OF DETECTION & LIMIT OF QUANTITATION

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 SYSTEM SUITABILITY
System suitability parameters are ;
Retention time,
Theoretical plates,
Asymmetry (Tailing factor).
Sample RT TP Asym. Area
Rep 1 4.58 8287 1.19 3512722
Rep 2 4.58 8365 1.10 3535254
Rep 3 4.58 8214 1.12 3525785
Rep 4 4.58 8202 1.09 3508674
Rep 5 4.58 8352 1.08 3518977
Rep 6 4.58 8288 1.15 3598741
Average 4.58 3533358.833
SD 9.72951E-16 33400.11779
%RSD 000 0.95
Table 7.14. System suitability data for SCG

 ACCURACY
The developed method was found to be accurate as
the % RSD values for accuracy studies were <2%,
as recommended by ICH guidelines.
Sr. no. Con.
(µg/ml)
Amt
Added
Area Amt
recov.
%
Recoveries
1 40 39.88 2808897 39.63 99.37
2 40 39.88 2813211 39.69 99.52
3 40 39.88 2827413 39.89 100.03
80% Mean 99.64
SD 0.342735077
%RSD 0.34
1 40 49.85 3512722 49.56 99.42
2 40 49.85 3535254 49.88 100.05
3 40 49.85 3525785 49.74 99.79
100% Mean (% Purity) 99.75
SD 0.3301959
%RSD 0.32
1 40 59.82 4223381 59.59 99.61
2 40 59.82 4259714 60.10 100.46
3 40 59.82 4258971 60.09 100.45
120% Mean 100.17
SD 0.48975366
%RSD 0.49
Fig. 7.13. HPLC chromatogram of SCG for 80%
Fig. 7.14. HPLC chromatogram of SCG for 120%
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Table 7.15. Accuracy data for SCG

 REPEATABILITY
A single sample was prepared as described and 6
injections were made from same sample and
checked for system suitability. It’s limit of %
RSD is <2 % as per ICH guidelines.
Sr.
No
Conc. (µg/ml) Area
1 40 3512722
2 40 3535254
3 40 3525785
4 40 3508674
5 40 3518977
6 40 3598741
AVG 3533358.833
SD 33400.11779
%RSD 0.95
Table 7.16. Repeatability data of SCG
1
2
3
Fig. 7.15. Repeatability chromatogram of SCG
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40
CHANNABASWESHWAR PHARMACY COLLEGE(DEGREE), LATUR

 ROBUSTNESS
Conditi
ons
Sample
ID
Area %
Assay
RT TP Asym.
0.8ml WS
DP
3687814
3652145
-
99.03
4.62
4.62
8324
8317
1.08
1.11
1.0ml WS
DP
3533358
3492674
-
98.85
4.58
4.58
8287
8178
1.09
1.15
1.2ml WS
DP
3692545
3659874
-
99.12
4.52
4.52
8237
8211
1.12
1.15
 Flow rate
WS : Working standard,
DP : Drug products
Table 7.17. Robustness data of SCG for different flow rates
0.8ml
1.0ml
1.2ml
Fig. 7.16. Chromatograms of SCG for different flow rates
7/13/2023
41

 Mobile phase composition :
Conditions
( % )
Sample
ID
Area %
Assay
RT TP Asym
metry
MP A = 78
MP B = 22
WS
DP
3529587
3502585
-
99.23
4.55
4.55
8345
8296
1.16
1.09
MP A = 80
MP B = 20
WS
DP
3533359
3492674
-
98.85
4.58
4.58
8287
8178
1.19
1.15
MP A = 82
MP B = 18
WS
DP
3578452
3556857
-
99.40
4.63
4.63
8421
8524
1.08
1.03
MP A = 0.1% Perchloric acid
MP B = Acetonitrile
Fig. 7.17. Chromatogram of SCG for MPA : MP B
78:22
80:20
82:18
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Table 7.18. Robustness data of SCG for different mobile phases

 SPECIFICITY
There is no interference of the blank solution in region
of the main peak of SCG . Hence the solvent system &
chromatographic conditions are specific for the
method.
Fig. 7.18. Chromatogram of blank solution of the solvent
 ASSAY
Label claim: Each ml of Eye drop contains contains 40
mg Sodium Cromoglycate. i.e. 0.125ml = 5mg of SCG.
0.125ml (1 drop) solution into 10ml volumetric flask and
make up volume to form 500µg/ml. solution. Pipette out 1ml
& make up to 10ml to form 50µg/ml final solution.
Formula:
% Assay = Sample area / Standard area ×100
Sample solution area = 3533358.83
Standard solution area = 3492674
% Assay = 98.85
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43

 ANALYSIS OF MARKETED FORMULATION
Table 7.19. Ophthalmic solution analysis data for % Assay
Sample ID RT Area % Assay
WS 4.59 3533359 -
DP 4.59 3492674 98.85
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44
Fig. 7.17. Chromatogram for drug product of the SCG

 SUMMARY
Parameters HPLC UV -Spectroscopy Limits
Range 40 - 60µg/ml 2 – 10µg/ml
(R2) 0.999 0.999 <1
Slope 69816.48 0.1744
Intercept 25055.2 0.112
Regression equation
(Y= mx + c)
Y=69816.48x + 25055.2 Y =0.1744x + 0.112
LOD 2.52µg/ml 0.05440 µg/ml To be determined
by test
LOQ 7.64µg/ml 0.1648 µg/ml
%Assay 98.85 % 101.4% 98 – 102%
System suitability 0.95 0.63940277 <2
Precision Repeatability -0.95% Intra-day1.789407
<2
Inter-day1.140092
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7/13/2023
46
Accuracy
(%recovery)
99.7% 100.05% 98 – 102%
Force degredation Photolytic 12.40 5-20%
Thermal 19
0.01N HCL 75.35
0.01N NaoH 77.26
H2O2
10% 21.43
20% 23.60
30% 26.53

 CONCLUSION
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47
 The developed UV-Visible & RP-HPLC method was found to be simple, accurate, rapid,
selective, precise & robust for estimation of drug in ophthalmic dosage forms.
 UV-Visible spectroscopy indicates that sodium cromoglycate is stable only in photolytic &
thermal environment not in acidic, basic &oxidative conditions.
 Validation was performed as per ICH- guidelines.

1. B. K. Sharma, Instrumental Methods of Chemical Analysis, Introduction to Analytical Chemistry, Goel
publishing house, Meerat, 10th edition, page no. 1 – 4, 200 – 203, 2000.
2. R. A. Nash and A. H. Wachter, Pharmaceutical Process Validation, an International third edition, Vol.
129.
3. CDER, Reviewer Guidance, “Validation of Chromatographic Methods.” 1994.
4. ICH Harmonized Tripartite Guideline. Validation of analytical procedure: Text and Methodology,
2005.
5. ICH, Q2A, Harmonized Tripartite Guideline, Validation of analytical Procedures Methodology,
IFPMA, Proceedings of the International Conference on Harmonization. Geneva, March 1994.
6. ICH Q2B, Harmonized Tripartite Guideline, Validation of analytical procedure methodology, IFPMA,
proceedings of the International Conference on Harmonization, Geneva, March 1996.
7. A. V. Kasture, S. G. Wadodkar, K. R. Mahadik, H. M. More, Pharmaceutical Analysis, 2nd edition, Vol.
1, page no. 1,4,48,169,1997.
8. Y. R. Sharma (2002), Elementary Organic Spectroscopy Principal and Chemical Analysis, S.Chand
publication, page no. 9.
9. G. R. Chatwal, S. R. Anand, Instrumental method of chemical analysis, Himalaya publication, 5th
edition, page no. 2.149, 2.566, 2.624.
 REFERENCES
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48

10. Paparaju.Sowjanya, Nissankararao.Srinath, Kamatham.Srinivas.Sumanth, Devarapalli.Chaitanya,
Ammula.Pushpa Sai, Vantakula.Padmaja. Validated RP-HPLC method for estimation of sodium
cromoglycate in human plasma. IJPRD, 2013; Vol 5(01): March-2013 (112 – 120) .
11. Maha A. Hegazy , Medhat A. Al-Ghobashy, Basma M. Eltanany and Fatma I. Khattab. Validated
chromatographic methods for the simultaneous determination of sodium cromoglycate and
oxymetazoline hydrochloride in a combined dosage form. Journal of Advances in Chemistry - 2015. Vol.
11, No. 8.
12. M. E. Fathy, S. Abo El Abass Mohamed, H. Elmansi, and F. Belal. Simultaneous determination of
cromolyn sodium combined dosage forms using isocratic HPLC method. Journal of Chromatographic
Science, 2017, Vol. 55, No. 1, 14–22.
13. El-Kosasy AM, Omar AA, Magdy N and El Zahar NM. Validated stability indicating HPLC method for
determination of the polyanionic cromolyn sodiumi in presence of its alkaline degradate. Analytical
Chemistry: An Indian Journal -2016, Vol-16, Iss – 13.
14. Maha A. Hegazy, May H. Abdelwahab, Hassan A. M. Hendawy, Soheir A.Weshahy & Samah S. Abbas.
Validated HPTLC and HPLC methods for determination of fluorometholone and sodium cromoglycate
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