pharmaceutical analysis

1. STABILITY INDICATING RP- HPLC METHOD DEVELOPMENT
& VALIDATION FOR THE SIMULTANEOUS ESTIMATION OF
LEVODROPROPIZINE AND CHLORPHENIRAMINE MALEATE
IN BULK AND THEIR PHARMACEUTICAL DOSGE FORM
Under the guidance of
Dr. K. Sujana, M.Pharm, Ph.D
(Pharmaceutical analysis)
1
Presented by:
T.Rajasekhar
M.Pharmacy
Y16MPH338

2. CONTENTS:
 Introduction
 Drug profile of selected drugs
 Literature review
 Aim & objective
 Plan of work
 Materials and Method
 Validation parameters results
 Stability indicating studies
 references
2

3. Introduction to chromatography
Chromatography:
It is a laboratory technique and physical method of separation mixture that distributes
components to separate between two phases, stationary phase and mobile phase, moving in a definite
direction.
Principle
 Partition coefficient
 adsorption
High Performance Liquid chromatography
HPLC is basically a highly improved form of column liquid chromatography. Instead of solvent
being allowed to drip through a column under gravity, it is forced through under high pressure of up to 400
atmospheres. That makes it must faster. All chromatographic separations, including HPLC operate under
same basic principle; separation of a sample into its constitution parts because of the difference in the
relative affinities of different molecules for the mobile phase and stationary phase used in the separation.
Types of HPLC
 Normal phase HPLC
 Reverse phase HPLC
 Size-exclusion HPLC
 Ion-Exchange HPLC
3

4. HPLC APPLICATION
Pharmaceutical application
1. To control drug stability
2. Pharmaceutical quality control
Environmental application
 Detection of phenolic compounds in drinking water
 Bio-monitoring of pollutants
Application in forensics
 Quantification of drugs in biological samples
 Identification of steroids in blood etc.
 Detection of cocaine and other drugs of abuse in blood, urine etc.
Food and flavour
 Measurement of Quality of soft drinks and water
 Sugar analysis in fruit juice
 Analysis of polycyclic compounds in vegetables
4

5. DRUG PROFILE
5

6. Drug profile of Levodropropizine
Sl.no Name of the parameter Description
1 IUPAC (2S)-3-(4-phenylpiperazin-1-yl)propane-1,2-diol
2 Molecular formula C13H20N2O2
3 Molecular weight 236.315 g/mol
4 Solubility slightly soluble in water
5 Boiling point 412.7°C
6 pKa Strongest Acidic 14
7 pKa Strongest Basic: 7.65
8 Terapeutic categery Cough suppressant
9 Storage Store in a tightly closed container at room
temperature. Protect from excess heat and moisture.
Keep out of reach of children. 6

7. Drug profile of Chlorpheniramine maleate
Sl.no Name of the parameter Description
1 IUPAC [3-(4-chlorophenyl)-3-(pyridin-2-
yl)propyl]dimethylamine
2 Molecular formula C16H19ClN2
3 Molecular weight 274.788 g/mole
4 solubility soluble in water
5 Boiling point 142 °C
6 Melting point 266 to 275° F
7 pKa: 9.13
8 storage Tightly closed containers
7

8. Marketed formulation
Sl.no Company
name
Brand
name
Strength Dosage form category
1 Dr.Reddys RESWAS 30 mg
levodropropzine,
2mg chlorpheniramine
Syrup Cough
suppresent
8

9. LITERATURE REVIEW
9

10. Sl.no TITLE solvents used λmax Results
1 1. Palakurthi Ashok KUMAR * 1, Thummala
Veera Raghava RAJU 1 , Dongala
THIRUPATHI
reverse phase-HPLC method has been developed
and validated for the simultaneous estimation of
Levodropropizine and methyle parabine in bulk
drug and pharmaceutical dosage form.
mobile phase of
Buffer: Acetonitrile
and orthophosphoric
buffer taken in the
ratio 45:55 v/v,
215nm . The retention times of
Levodropropizine and
chlorophrnraminei were 2.124
min and 3.334 min
respectively.r²of
Levodropropizine0.999% and
chloropheniramineis 0.999%,LOD
and LOQ of levodropropizineis
0.15 and 0.46 methyle parabinis
0.28 and 0.84
2 ZHANG Qing1,LIU Jun1*,HUANG
Fang2,ZHOU Yi-bin2
RP-HPLC method has been developed for the
validated of Levodropropizine and Benzopine in
bulk and combined Tablet dosage form
Acetonitrile: 0.1%
perchloric acid (50:50
v/v) as the mobile
phase
230 nm The retention time of the
levodropropizine and
Benzopine4.25, 6.05 min
respectively.r² of
Levodropropizine is 0.999%
Benzopine is 0.999%
Lod of levodropropizine is 0.17
loq is 0.42 Benzopine is 0.52 and
0.26
3 Lin Yan*,Tongling LiRongqin, ZhangXiaohong
Xu,Pengcheng Zheng.et al.,(2005).
Stability indicating RP-HPLC method was
developed for the quantitation of
Levodropropizine in its pharmaceutical dosage
form
0.01N potassium
dihydrogen phosphate
buffer and acetonitrile
(50:50%v/v) as mobile
phase on isocratic
mode
240nm. The retention time for
Levodropropizine were found to
be 2.32min a respectively lod is
0.26 and loq is 0.17
Levodropropizine is 0.78
10

11. Aim & Objective
Aim
The main aim of the present study is to develop an accurate, precise, sensitive, selective, reproducible
and rapid analytical technique for simultaneous estimation of Levodropropizine, Chlorpheniramine in
bulk and Syrup dosage form.
Objective and Plan:
 Following are the objectives of the present work:
 To develop a new stability indicating HPLC method for simultaneous estimation of
Levodropropizine and Chlorpheniramine and to develop the validated method according to ICH
guidelines.
 To apply the validated method for the simultaneous estimation of Levodropropizine and
Chlorpheniramine in pharmaceutical formulation
11

12. Plan of work
Plan of work for RP-HPLC was as follows:
• From the UV-analysis selection of λmax. Selection of chromatographic
condition.
• Selection of suitable solvent.
• Selection of stationary phase.
• Selection of mobile phase.
• Selection of flow rate.
• Selection of initial separation condition.
• Optimization of chromatographic and spectral conditions.
• Validation of proposed method.
• Designing of forced Degradation studies and Separation of formed
Degradative products.
12

13. Materials and Method
• Materials:
Levodropropizine(LZD) and Chlorpheniramine(CPM) pure drugs (API), Combination
Levodropropizine and Chlorpheniramine syrup (RESWAS), Distilled water, Acetonitrile,
Phosphate buffer, , Methanol, Potassium dehydrogenate ortho phosphate buffer, Ortho-
phosphoric acid. All the above chemicals and solvents are from Rankem
• Instruments:
• Electronics Balance-Denver
• pH meter -BVK enterprises, India
• Ultrasonicator-BVK enterprises
• WATERS HPLC 2695 SYSTEM equipped with quaternary pumps, Photo Diode Array
detector and Auto sampler integrated with Empower 2 Software.
• UV-VIS spectrophotometer PG Instruments T60 with special bandwidth of 2 mm and
10mm and matched quartz cells integrated with UV win 6 Software was used for
measuring absorbances of Levodropropazine and Chloropheniramine solutions.
13

14. Standerd and Sample Preparation
• Diluent: Based up on the solubility of the drugs, diluent was selected, Acetonitrile and Water taken in the ratio of
50:50
• Preparation of Standard stock solutions: Accurately weighed 30mg of Levodropropizine, 2mg of
Chlorpheniramine and transferred to individual 50 ml volumetric flasks separately. 3/4 th of diluents was added to
both of these flasks and sonicated for 10 minutes. Flasks were made up with diluents and labeled as Standard stock
solution 1and 2. (600µg/ml of Levodropropizine and 40µg/ml of Chlorpheniramine )
• Preparation of Standard working solutions (100% solution): 1ml from each stock solution was pipetted out and
taken into a 10ml volumetric flask and made up with diluent. (60µg/ml Levodropropizine of and 4µg/ml of
Chlorpheniramine )
• Preparation of Sample stock solutions: Syrup equivalent to 30mg Levodropropizine and 2mg of Chlorpheniramine
was transferred into a 50 ml volumetric flask, 20ml of diluents was added and sonicated for 25min, further the volume
was made up with diluent and filtered by HPLC filters (600µg/ml of Levodropropizine and 40µg/ml of
Chlorpheniramine )
• Preparation of Sample working solutions (100% solution): 1ml of filtered sample stock solution was transferred to
10ml volumetric flask and made up with diluent. (60µg/ml of Levodropropizine and 4µg/ml of Chlorpheniramine )
• Preparation of buffer:
0.1% OPABuffer: 1ml of Ortho Phosphoric acid was diluted to 1000ml with water.
14

15. Chromatographic Trails
Trail 1 of Levodropropizine and chlorpheniramine maleate
15
Column Kromosil C18(4.6×250nm, 5µm
Flow rate 1 mL /min
Wavelength 229nm
Column temperature 30°C
Injection volume 20 µL
Run time 10 minutes
Mobile phase Water : Methanol (50%:50%)
Remarks Both were eluted but peak shape is not good so, further trial is carried out

16. • Trail 2
16
Column Azilent C18 (4.6 x 150mm, 5µm)
Flow rate 1 mL /min
Wavelength 215nm
Column temperature 30°C
Injection volume 10 µL
Run time 10 minutes
Mobile phase Water and Acetonitrile in the ratio 50%:50%
Remarks
Both peaks were eluted but peak shape, USP plate count &
tailing factor were not good So, Further Trial is carried out

17. • Trail 3
17
Column Azilent C18 (4.6 x 150mm, 5µm)
Flow rate 1 mL /min
Wavelength 215nm
Column temperature 30°C
Injection volume 10 µL
Run time 10 minutes
Mobile phase Water and Acetonitrile in the ratio 50%:50%
Remarks
Both peaks were eluted, peak shape was not good &
Levodropropizine eluted at void time So, further trial is Carried out

18. • Trail 4
18
Column Azilent C18 (4.6 x 150mm, 5µm)
Flow rate 1 mL /min
Wavelength 215nm
Column temperature 30°C
Injection volume 10 µL
Run time 10 minutes
Mobile phase Water and Acetonitrile in the ratio 50%:50%
Remarks
Both peaks were eluted, peak shape was not good &
Levodropropizine eluted at void time So, further trial is Carried out

19. • Trail 5
19
Column Azilent C18 (4.6 x 150mm, 5µm)
Flow rate 0.8mL /min
Wavelength 215nm
Column temperature 30°C
Injection volume 20 µL
Run time 10 minutes
Mobile phase Water: Acetonitrile(50%:50%)
Remarks In this trail both peaks shape were good but retention time was
more so, further trial is carried out

20. • Optimised chromatogram LZD & CPM
20
Column Kromosil C18 (4.6 x 250mm, 5µm)
Flow rate 1ml/min
Wavelength 215nm
Column temperature 30°C
Injection volume 10 µL
Run time 7 minutes
Mobile phase 45% 0.1% OPA: 55% Acetonitrile
Remarks Both peaks have good resolution, tailing factor,
Theoretical plate count and resolution

21. Assay results of levodrpropizine and chlorpheniramine
21
Name Labelled amount of
drug (mg)
Mean amount found by the
proposed method
(n=6)
% Assay
Levodropro and
chlorpheniramine
30(mg)
2(mg)
29.99
1.99
99.61
99.77
typical chromatogram of levodropropizine and
chloropheniramine(Formulation)

22. Validation parameters for analytical method
 The objective of validation of an analytical method is to demonstrate that it is
suitable for its intended purpose.
 Validation of analytical methods is the process by which it is established by
laboratory studies that the performance characteristics of the method meet the
established requirements for the analytical applications. According to ICH,
typical analytical performance characteristics that should be considered in the
validation of all the types of methods are:
A) Accuracy
B) Precision
C) Specificity
D) Limit of detection
E) Limit of quantification
F) Range
G) Robustness
H) Linearity
I) Ruggedness
j) sensitivity
k) System suitability
22

23. System suitability parameter
23
Parameter Results
Tailing factor
Resolution
Plate count
LEVODROPRO CHLORPHENI
1.38
3143
1.23
7.5
4270

24. Linearity:
Linearity table for Levodropropazine and Chloropheniramine
24
Levodropropazine Chloropheniramine
Conc (μg/mL) Peak area Conc (μg/mL) Peak area
0 0 0 0
15 392446 1 95526
30 803281 2 180885
45 1192608 3 258447
60 1535084 4 347067
75 1909233 5 430020
90 2282051 6 516857
y = 25266x + 22293
R² = 0.9994
0
500000
1000000
1500000
2000000
2500000
0 20 40 60 80 100
y = 85205x + 5642.1
R² = 0.9996
0
100000
200000
300000
400000
500000
600000
0 1 2 3 4 5 6 7

25. Parameter Results
levodropropizine Chloropheniramine
Linearity range
Correlation coefficient
Slope
Y-intercept
15-90µg/ml
0.999
25266
22293
1-6µg/ml
0.999
85205
5642
25
Linearity results

26. 26
Drugs
%Concentration
(at specification
Level)
Area
Amount
Added
(mg)
Amount
Found
(mg)
% Recovery
Mean
Recovery
(n=3)
LZD
50% 225703.3 46 46.32 100.69
100.39100% 448469.7 92 92.03 100.04
150% 675482.7 138 138.62 100.45
CPM
50% 109553.3 7.5 7.53 100.44
100.39100% 219228.7 15 15.07 100.50
150% 327988.3 22.5 22.55 100.24
ACCURACY

27. Precision
System precision
27
S. No
Area of
Levodropropizine
Area of
Chlorpheniramine
1. 1543394 343899
2. 1555298 346789
3. 1550708 347148
4. 1539671 349348
5. 1542082 348735
6. 1540428 349450
Mean 1545264 347562
S.D 6302.5 2112.7
%RSD 0.4 0.6
S. No
Area of
Levodropropazine
Area of
Chloropheniramine
1. 1521591 339702
2. 1526990 338651
3. 1529710 338908
4. 1516533 337858
5. 1513665 339480
6. 1529088 334999
Mean 1522930 338266
S.D 6766.5 1728.4
%RS
D 0.4 0.5
Repeatability

28. 28
S.No Area of
LZD(Day1)
Area of LZD
(day2)
Area of
CPM(Day1)
Area of
CPM(Day2)
1 1532352 1532351
345667 345667
2 1539915 1539911
347408 347408
3 1538104 1538103
346394 346394
4
1537173 1537173 345820 345820
5
1539452 1539452 344647 344647
6
1535326 1535326 347739 347739
Mean
1537054 1537054 346279 346279
S.d
2833.6 2833.6 1154.8 1154.8
%RSD
0.2 0.2 0.3 0.3
Intermediate precision day to day

29. S.
n
o
Condition area count
of LZD
Area of CPM Rt of
LZD
Rt of
CPM
S.D of
LZD
S.D of CPM %RSD of
LZD
%RSD of
CPM
1 Flow rate decrease
0.9ml/min
1714240 388128 2.48 4.091 4030 2027 0.2 0.5
2 Flow rate
increase1.1ml/min
1549848 351272 2.24 3.54 4066 2403 0.3 0.7
3 Mobile phase
decrese 45B:55A
15670779 359501 2.24 3.56 3926 1987 0.1 1.1
4 Mobile phase
increase 35B:65A
1598041 370712 2.33 4.01 11123 5981 0.7 1.6
5 Temperature
decrese 25°C
1659688 373739 2.32 3.94 10842 719 0.7 0.2
6 Temperature
increase 35°C
162986 37296 2.24 3.59 13043 3635 0.8 0.9
29
ROBUSTNESS

30. S.no Name RT(min) Area USP
Tailing
USP Platecount Resolution
1 LZN 2.26 1549848 1.30 3592
2 CPM 3.76 355241 1.25 5581 7.1
Specificity
30

31. S.NO PARAMETER Levodropropizine Chlorpheniramine
1 LOD 0.39µg /ml 1.18µg /ml
2 LOQ 0.06µg /ml 0.19µg /ml
31
LOD&LOQ:

32. Parameter Results
Levodropropizine Chloropheniramine
λ max 215nm 215 nm
correlation coefficient 0.999 0.999
Beer’s limit 15-100µg/ml 1-6µg/ml
Slope 25266 85205
Intercept 22293 5642
Accuracy 100.39%Recovery 100.39%Recovery
Precision 0.4% RSD 0.6% RSD
Robustness 0.2%RSD 0.3%RSD
LOD 0.39 µg /ml 1.18µg /ml
LOQ 0.06µg /ml 0.19µg /ml
Summary of validation parameters
32

33. FORCED DEGRADATION STUDIES
Acid degradation: 2 ml of stock solution of Levodropropzine and Chlorpheniramine were
taken, and 1ml of 1N Hydrochloric acid was added and kept at 600c for 24 hrs. Then the
solution was neutralized with 1ml of 1N Sodium hydroxide. The resultant solution was
diluted with diluent and 20 µl solutions were injected into the system and chromatogram
was recorded.
Base degradation: 2 ml of stock solution of Levodropropizine and Chlorpheniramine were
taken, and 1 ml of 1N sodium hydroxide was added and kept at 60⁰c for 24hrs. Then the
solution was neutralized with 1 ml of 1N Hydrochloric acid. The resultant solution was
diluted with diluent and 20 µl were injected into the system chromatogram was recorded .
Peroxide degradation: 2ml of stock solution of Levodropropizine and Chlorpheniramine
were taken, and 3 ml of 30 % Hydrogen peroxide was added and kept at 60⁰c for 24 hrs.
The resultant solution was diluted with diluent. and 20 µl were injected into the system and
chromatogram was recorded.
Thermal degradation: 2 ml of stock solution of levodropropizine and Chlorpheniramine
were taken and placed in oven at 600c for 4hrs to study dry heat degradation For HPLC
study, the resultant solution was diluted with diluent and 20µl were injected into the system
and the chromatogram was recorded.
Photo degradation: 2 ml of stock solution of Levodropropizine and Chlorpheniramin
ewere taken and placed in sunlight for 24hrs For HPLC study, the resultant solution was
diluted with diluent solution and 20µl were injected into the system and the chromatogram
was recorded .
33

34. Acid degradation peak Base degradation peak
peroxide degradation peak Thermal degradation peak
34

35. 35
Uv degradation peak Water degradation peak

36. Type of
degradati
on
Levodropropazine Chloropheniramine
AREA %RECOV
ERED
%
DEGRA
DED
AREA %RECOVER
ED
% DEGRADED
Acid
1463168 94.59 5.41 330149 94.90 5.10
Base
1485270 96.02 3.98 332523 95.58 4.42
Peroxide
1495245 96.67 3.33 335361 96.39 3.61
Thermal
1505719 97.34 2.66 340862 97.97 2.03
Uv
1521308 98.35 1.65 341484 98.15 1.85
Water
1531908 98.35 1.65 344931 99.14 0.86
Summary of degradation
36

37. CONCLUSION:
The present work concluded that stability indicating assay method by RP-HPLC was
simple, accurate, precise, and specific and has no interference with the placebo and
degradation products. Hence these can be used for routine analysis of Levodropropizine and
Chlorpheniramine malete.
The developed HPLC method for the estimation of selected drugs is simple, rapid,
accurate, precise, robust and economical. The mobile phase and solvents are simple to
prepare and economical, reliable, sensitive and less time consuming.
37

38. ACKNOWLEDGMENT
I would like to thankful Prof. A. Prameela Rani, M.Pharm,Ph.D
I would like thankful to my guide Dr.Sujana M.Pharm,Ph.d
38

39. • References
• MacNair J.E., Patel K.D. And Jorgenson J.W., Ultra pressure reversed phase capillary
Liquid Chromatography: Isocratic and gradient elusion using columns packed with
1μm particles, Anal. Chem. 1999; 71: 700–708.
• Wu N., Lippert J.A. And Lee M.L., "Practical aspects of ultrahigh pressure capillary
liquid chromatography,, J. Chromatogr., 2001; A 911: 1–12.
• Swartz M. E. , Ultra Performance Liquid Chromatography (UPLC): An Introduction,
Separation Science Re-Defined, LCGC Supplement, p. 8-11(MAY 2005).
• Jerkovich A.D., Mellors J.S., and Jorgenson J.W., The use of micrometer-sized particles
in ultrahigh pressure liquid chromatography. LCGC. 2003; 21(7): 600–610.
• MacNair J.E., Lewis K.C. And Jorgenson J.W., Ultrahigh-pressure reversed-phase
liquid chromatography in packed capillary columns. Anal. Chem. 1997; 69: 983–989.
• Van Deemter JJ, Zuiderweg EJ, Klinkenberg A.Longitudinal diffusion and resistance to
mass transfer as causes of non ideality in chromatography. Chem. Eng. Sci. 1956; 5:
271–289.
39

40. • Goodwin L, White SA, Spooner N. Evaluation of ultra-performance liquid
chromatography in the bioanalysis of small molecule drug candidates in plasma.J.
Chromatogr. Sci. 2007; 45(6): 298–304.
• Swartz M., Ultra Performance Liquid Chromatography. LCGC. 2005; 23(1): 46–53.
• Broske A.D., et al., Agilent Technologies application note 5988-9251EN (2004).
• Michael E. Swartz and Brian J. Murphy. Ultra performance liquid chromatography:
tomorrow's HPLC technology today as published in LPI- June 2004.
• Michael E. Swartz, UPLC: An Introduction and Review Waters Corporation, Milford,
Massachusetts, USA, Journal of Liquid Chromatography & Related Technologies, 2005;
28: 1253–1263.
• Gui-Ling Yang, Li-Wei Yang2, Yong-Xue Li, Hui Cao,Wen-Liang Zhou, Zhi-Jian Fang,
et al. Applications of Ultra-Performance Liquid Chromatography to Traditional Chinese
Medicines. J.Chromatogr Sci., 2008; 48: 18-21.
• https://www.scbt.com › ... › Inhibitors, Activators, Substrates › Protein › PK
• https://www.drugbank.ca/drugs/DB01114
• https://www.drugbank.ca/drugs/DB12472
• https://www.scbt.com › Home › Chemicals › Other Chemicals › Antioxidants
40

41. 41