DEVELOPMENT AND VALIDATION OF STABILITY INDICATING ANALYTICAL METHOD FOR THE ESTIMATION OF MIDODRINE HYDROCHLORIDE ORAL SOLUTION BY RP HPLC METHOD

1. “DEVELOPMENT AND VALIDATION OF
STABILITY INDICATING ANALYTICAL
METHOD FOR THE ESTIMATION OF
MIDODRINE HYDROCHLORIDE ORAL
SOLUTION BY RP HPLC
Presented By
Mr. Kanade Sagar A.
M.Pharm II yr.
Department of Pharmaceutical Chemistry
S.R.T.M.U. Nanded.
Guided by:
Dr. S.S. Pekamwar
Professor, SOP,
Department of Pharmaceutical Chemistry
S.R.T.M.U. Nanded.

2. CONTENTS
1. INTRODUCTION
2. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
3. INSTRUMENTATION
4. APPLICATIONS OF HPLC
5. REVIEW OF LITERATURE
6. AIM & OBJECTIVES
7. PLAN OF WORK
8. WORK DONE
9. DRUG PROFILE
10. METHOD DEVELOPMENT
11. VALIDATION OF METHOD
12. REFERENCES

3. INTRODUCTION:-
 The Analytical chemistry may be defined as the science and
art of developing accurate, precise and sensitive methods
for determining the composition of materials in terms of
elements or compounds contained.
 METHOD DEVELOPMENT
 Method development is a challenging and very much a trial-
and-error approach.
 CONSIDERATIONS BEFORE METHOD DEVELOPMENT
 Before starting the arduous process, a thorough literature
search should be conducted for existing methodologies.
 It often provides a starting point for method development.3

4. NEW ANALYTICAL METHODS ARE NEEDED FOR THE
FOLLOWING REASONS
Existing methods are not available.
Existing methods are not sufficiently reliable, sensitive, or
cost effective.
New instrumentation or technique has better performance.
An alternate method is required for regulatory compliance.
PRINCIPLE OF CHROMATOGRAPHY
It is the method used primarily for the separation of the
components of a sample, in which the components are
distributed between two phases, one of which is stationary
while the other is mobile.
4

5. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
Same principle of separation as classical column
chromatography but here mobile phase is pumped through
the packed column under high pressure.
METHOD DEVELOPMENT IN HPLC
Before proceeding with development of method for a
particular sample it is absolutely essential to have detailed
information about the sample and separation goal should be
clearly defined.
5

6. METHOD DEVELOPMENT :
 Today the development of a method of analysis is usually
based on prior art or existing literature, using the same or
quite similar instrumentation. Method development usually
requires selecting the method requirement and deciding on
what type of instrumentation to utilize and why.
 Reason for developing new method of analysis is:
 There may not be suitable method for particular analyte in the
specific sample matrix.
 Existing method may be inaccurate, artifact, or contamination
prone or they may unreliable.
 Existing method may be too expensive, time consuming, or
energy intensive, or they may not be easily automated.
 Newer instrumentation and technique may have evolved that
provide opportunities for improved method.
6

7. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
 This technique is based on the same method of separation as classical column
chromatography.
 It is the most popular technique today among the different chromatographic
procedures.
 The development of HPLC has enabled LC to achieve great success in providing
following features.

8. 1. Speed of separation
2. High resolving power
3. Monitoring the column effluent
4. Repetitive and reproducible analysis.
5. Automation of analytical procedure
Cont

9. CONT..
 There are two type of HPLC depending upon nature of stationary
phase and mobile phase.
1. Normal phase chromatography
2.Reverse phase chromatography
 Normal phase HPLC has a polar stationary phase and non- polar
mobile phase
 Reversed phase HPLC (RP-HPLC or RPC) has a non-polar
stationary phase and an aqueous, moderately polar mobile phase
 One common stationary phase is silica.

10. INSTRUMENTATION
Parts of RP-HPLC
1. Solvent Reservoir
2. Degasser
3. High Pressure Pump
4. Sample Injection Port
5. Column
6. Detector
7. Data Processing and
Control
8. Waste

11. APPLICATIONS OF HPLC
 Qualitative Analysis:
a. HPLC is used for identification of compound.
b. It is used to check the purity of compound.
c. It is also used to check the presence of impurity in compound.
d. If the impurity presence in the sample we observed additional
peak when we compared the chromatogram of test with the
reference standard.
 Quantitative Analysis:
a. HPLC used for perform the assay of many compound.
b. It is used for determination of drug mixture.
c. Biopharmaceutical and pharmacokinetic study.
11

12. CONT..
d. It used for stability study.
e. Also used for purification of compound.
f. Investigation of biological material e.g. Blood, Urine.
g. Many poisonous substances can also be investigated by
HPLC
 Biochemical Analysis:
E.g. Carbohydrate, Lipid, Vitamin, Amino Acid.

13. REVIEW OF LITERATURE (5-11):
The literature survey shows that some methods are developed and
validated for the analysis of Anti-Hypotensive drug.
1] Hemant K. Jain* et al; (2017) Hemant K. Jain et al; (2017) had
reported a formulation development and stability indicating HPLC
assay of tablets of Apixaban.
Mobile Phase Water: Acetonitrile (60:40) v/v+0.1 % TEA
Column Purospher Star RP-18 end-capped (5 μm) Hibar 250x4, 6
Detector UV Detector
λ max 280nm

14. 2] Hemant K. Jain et al; (2017) had development and
validation of HPTLC method for determination Apixaban in
bulk and tablets.
Mobile Phase Toluene, Methanol and Diethylamine
( 16:3:1 v/v/v).
TLC Plate Al plates (10x10 cm) Si 60 F254
λ max 288nm

15. CONT..
3] Hemant K. Jain* et al; (2016) had reported a
development of an UV spectrophotometric method for
estimation of Midodrine hydrochloride in bulk and
tablets.
Detector UV Detector
λ Max 302nm

16. CONT..
4] Hemant K. Jain et al; (2016) had reported a
development of an accurate, simple, sensitive and precise
stability indicating reverse phase-high performance liquid
chromatographic (RPHPLC) assay method for estimation
of Midodrine hydrochloride in bulk and tablets.
Mobile Phase Triethylamine buffer 0.02%, pH-3: Acetonitrile
(38:62 v/v)
Column C18, (250 mm X 4.6 mm, 5 μm)
Detector UV Detector
λ max 289nm

17. CONT..
5] Dr. Mrinalini C. Damle et al; (2016) had reported a
simple and rapid stability indicating HPTLC method for
Midodrine hydrochloride was successfully developed.
Mobile Phase n-butanol: Methanol: water (6:2:2 v/v/v)
TLC Plate Al sheets precoated with silica gel 60F254
λ max 290nm

18. CONT..
6] Pritam S. Jain* et al; (2015) had reported Area
under Curve Method Development and Validation
of Midodrine Hydrocholride
Detector UV Detector
λ Max 278 and 299 nm

19. CONT..
7] Mamdouh R.Rezk * et al; (2015) had reported
Selective determination of midodrine hydrochloride in
the presence of its acidic degradation product.
Mobile Phase 0.05M PotassiumDihydrogen Phosphate:
Acetonitrile (80:20, V/V)
Column Zorbax C18 column (250mm
× 4.6 mm; 5 ìm).
λ max 290nm

20. AIM & OBJECTIVES
DEVELOPMENT AND VALIDATION OF STABILITY
INDICATING RP-HPLC METHOD FOR ESTIMATION OF
MIDODRINE HYDROCHLORIDE ORAL SOLUTION.
To validate the developed method as per ICH guidelines.
To study stability of drug under stress condition.(Force degradation
study)

21. PLAN OF WORK-
A. Literature survey
B. Selection of Drug molecules
C. Selection of mobile phase
D. selection of column
E. Optimization of chromatographic condition.
F System suitability parameter
G. Validation parameter
21

22. DRUG PROFILE-
Drug
name
MIDODRINE HYDROCHLORIDE
Structure
IUPAC name (±)-1-(2, 5-Dimethoxyphenyl)-2-glycinamidoethanol
22

23. Molecular formula C12H18N2O4. HCL
Molecular weight 290.74g/mol
pKa 7.8 (0.3% aqueous
solution
Appearance White to pale yellow
crystalline powder.
Category Anti-Hypotensive
Half life 4 hours
Melting point 200-203 °C
Solubility Soluble in water, sparingly
soluble in methanol
23

24.  Mode of action: Midodrine hydrochloride is a prodrug which
forms an active metabolite, desglymidodrine, which is an α1-
receptor agonist and exerts its actions via activation of
the alpha-adrenergic receptors of the arteriolar and venous
vasculature, producing an increase in vascular tone and
elevation of blood pressure.
ADR: Headache; feeling of pressure/fullness in the head,
vasodilation/flushing face,
confusion/thinking abnormality,
dry mouth; nervousness/anxiety and rash.

25. I. Validation Proposed Method
For assay
1.Accuracy & Recovery Study
2.Pricision
3. Linearity and Range
4.Rubustness
5.Specificity And Selectivity
II. Forced degradation study:-
1.Temperature 4. Oxidation
2.Acid Hydrolysis 5.Light (UV/Vis/Fl)
3.Base Hydrolysis 25

26. WORK DONE-
Identification of Drug-
UV
26
Wavelength Absorban
ce
290 0.5816

27. FTIR
Standard IR Ranges
(cm-1)
IR Ranges
(cm-1)
Functional Group
3400-3700 3327 O-H Acohol
2850-2950 2876 C-H ALKANE
1080-1360 1296 C-N STRECH
1400-1600 1484 C=C STRECH OF AROMATIC RING
1000-1300 1162 C-O STRECH
675-1000 854 C-H BENDING OF BENZENE
27

28. OPTIMIZATION OF CHROMATOGRAPHIC CONDITION-
 Selection of mobile phase-
28
Mobile Phase Proportion Flow rate Rt
Buffer pH- 4.0:
Acetonitrile
88:12 1.0 ml/min 8.0Min
Buffer pH- 4.0:
Acetonitrile
88:12 1.0 ml/min 7.3min
Buffer pH- 4.0 :
Acetonitrile
88:12 1.0 ml/min 7.0min
Buffer pH- 4.0:
Acetonitrile
88:12 1.2 ml/min 4.0min

29. 29
Column : GL-Science, Inertsil ODS 3V
C18, 5µ, 4.6 x 150mm
Flow Rate : 1.0 mL/min
Injection Volume : 20 µL
Wavelength : 290 nm
Column Temp : Ambient
Sample Temp : 10ºC
Run Time : 12.0 minutes
Retention Time : Midodrine Hydrochloride
peak about 3.7 minutes
Seal wash : Water: Acetonitrile(90:10) v/v
Needle wash : Water: Acetonitrile(10:90) v/v
Optimized Chromatographic Condition-

31. Buffer pH-4.0: Acetonitrile 88:12
Conclusion:-midodrine peak observed at 8.09 min,
however as the solution contain preservative they are not
observed in this method at 290.0 nm nor at 254nm method
needs to be optimised
0.015M Without TEA
31

32. Buffer pH-4.0 : Acetonitrile 88:12
Conclusion :-midodrine peak observe at 7.07 min,however as
the solution contain preservative they are not observed in this
method at 290.0 nm nor at 254nm
0.015M MP-B WITH TEA
32

33. MOBILE PHASE A-BUFFER PH-4 AND MOBILE PHASE-B- ACN
Conclusion :-
Midodrine peak observed at 4.002min at 290nm at the same time
methyl paraben and propyl paraben peak observe at 6 and 7 min at
254nm method is specific both midodrine preservative.
STD Chromatograph
33

34. MOBILE PHASE A-BUFFER PH-4 AND MOBILE PHASE-B-
ACN
34
Midodrine Sample Chromatograph
Conclusion :-
Midodrine peak observed at 4.014min at 290nm at the same time
methyl paraben and propyl paraben peak observe at 6 and 7 min at
254nm method is specific both midodrine preservative.

35. BLANK AND PLACEBO
35
Conclusion :-
Midodrine placebo peak observed at 7.2min and 8.1min at
290nm at the both midodrine preservative.

36. EXPERIMENTAL AND RESULTS.
36
 2] Marketed formulation & API
 1. Working Standard / Drug Sample
 The gift sample was obtained 1) Midodrine
hydrochloride from ENALTEC Pharma Research
Pvt. Ltd. Thane, Maharashtra.

 2. Formulation
 Midodrine hydrochloride ORAL SOLUTION Mfg. by
ENALTEC Pharma Research Pvt. Ltd. Thane,
Maharashtra

37. . CHEMICALS AND REAGENTS
Sr. No.
Chemicals / Materials Make Grade / Batch No
1 Reagent Name Grade Make
2 Water HPLC -
3 Acetonitrile HPLC Rankem or equivalent
4 Potassium dihydrogen
orthophosphate
AR Merck or equivalent
5 Phosphoric acid 88% Analysis Merck or equivalent
6 0.45µ PVDF Syringe Filter Millipore Millex HV or equivalent
7 0.45µ Nylon membrane
disc Filter
Ultipor® PALL Life science or
equivalent
37

38. 38
 REAGENTS AND MATERIALS USED:
 Water (Milli Q or equivalent)
 Acetonitrile (HPLC Gradient Grade, Rankem)
 Methanol (HPLC Gradient Grade, Merck)
 Triethyl amine (For Chromatography, Merck)
 Ortho-phosphoric acid (GR Grade, Merck)
 0.45µ Teflon + Glass membrane filter (Make: mdi-
Cat.No.SYTG0602MNXX104) or equivalent
 INSTRUMENT USED:
. Instruments
1. Digital pH Meter: Make: Thermo scientific; Model: Orian Star A211
2. Analytical weighing balance: Make: Mettler Toledo; Model: XS205D0.
[Max. 80g, Min. 20mg; ]
3. HPLC [ Make: Waters; Model No-2695; Uv]
4. Sonicator- Ultrasonic Bath

39. VALIDATION OF METHOD
 Method validation is the process used to confirm that the
analytical procedure employed for a specific test is suitable for
its intended use.
 Results from method validation can be used to judge the
quality, reliability and consistency of analytical results; it is an
integral part of any good analytical practice.
 The USP has published specific guidelines for method
validation for compound evaluation.

40. 40
SYSTEM SUITABILITY TEST:
PREPARATION OF STD. SOLUTION :
Weigh accurately about 25 mg of Midodrine Hydrochloride standard in 100
mL volumetric flask. Add 70 mL of diluent, sonicate to dissolve and dilute
up to the mark with diluent.
(Concentration of Midodrine Hydrochloride standard solution: 0.25mg/ml)
Component
Midodrine
Hydrochloride
USP Tailing 1.2
Theoretical Plates 3481
S. No. Area
1 3373600
2 3378530
3 3384508
4 3375913
5 3375999
Mean 3377710
%RSD 0.12
Correlation 99.8

41. 41
APPLICATION OF PROPOSED METHOD FOR ESTIMATION OF
PREPARATION OF SAMPLE SOLUTION :
Fig: chromatogram of standard solution of atazanavir sulfate

42. 42
VALIDATION PARAMETERS:
SPECIFICITY:-
Fig 13: chromatogram of placebo preparation
Fig 13:
chromatogram of
blank preparation
Fig 13: chromatogram of std. preparation

43. 43
Fig 20: chromatogram of sample
preparation Fig 20: chromatogram of spiked sample preparation

44. 44
Specificity

45. 45
C) FORCED DEGRADATION STUDIES
Fig 21: chromatogram of control sample.
Fig 22: chromatogram of acid degradation sample.

46. 46
Fig 22: chromatogram of acid degradation sample.

47. 47
Fig 23: chromatogram of base degradation sample.
Fig 23: chromatogram of base degradation placebo.

48. 48
Fig 24: chromatogram of peroxide degradation sample.
Fig 24: chromatogram of peroxide degradation placebo.

49. 49
Fig 25: chromatogram of Degradation of Midodrine hcl spl open&close
.
Fig 25: chromatogram of Degradation of Midodrine hcl placebo open&close
.

50. 50
Conditions % Assay
Degradation
achieved
Purity
angle
Purity
threshold
Control 97.2 0.027 0.226
Acid degradation 92.1 5.1 0.009 0.259
Base degradation 87.9 9.3 0.160 0.262
Peroxide degradation 90.1 7.1 0.069 0.501
Thermal degradation 95.3 1.9 0.092 0.354

51. 51
LINEARITY:
Level (%) Concentration (ppm)
Response
1 2 Mean
50 0.125 1683834 1680964 1682399
80 0.187 2501958 2510739 2506349
100 0.250 3330530 3334056 3332293
120 0.312 4180336 4170727 4175532
150 0.375 5011115 5004008 5007562
Co-relation coefficient (r2) 1.000
SLOPE 13324.816
Y-INTERCEPT 13018.052
WORKING LEVEL AREA 3332293
%LIMIT OF Y-INTERCEPT ( ± 5 OF WORKING LEVEL) 0.39
y = 13,324.816x + 13,018.052
R² = 1.000
0
1000000
2000000
3000000
4000000
5000000
6000000
0 50 100 150 200 250 300 350 400
Area
Concentration mcg/mL
MIDODRINE HCL

52. 52
ACCURACY:
PRECISION:
SYSTEM PRECISION:
Level (%)V
Theoretical
concentration (mcg/mL)
% Recovery
Mean
recovery
%
50
125.828 100.7
100.5125.367 100.3
125.826 100.6
100
249.958 100.0
100.1250.564 100.2
250.644 100.2
150
377.539 100.7
100.7377.176 100.6
378.431 100.9
Mean recovery 100.5
%RSD 0.17
Tailing factor 1.2
Theoretical plates 3481
Sr. No. Area
1 3373600
2 3378530
3 3384508
4 3375913
5 3375999
Mean 3377710
%RSD 0.12

53. 53
METHOD PRECISION: INTERMEDIATE PRECISION:
The pooled data obtained from Analyst-I and II is summarized in below
Tailing factor 1.2
Theoretical plates 3481
S. No. Area
1 3373600
2 3378530
3 3384508
4 3375913
5 3375999
6 3377710
Mean 3384490.667
%RSD 0.12
Tailing factor 2.0
Theoretical plates 7390
Sr. No. Area
1 3418232
2 3418542
3 3418351
4 3410611
5 3416973
Mean 3416541.8
% RSD 0.10
Parameter Method Precision (Analyst-I)
Intermediate Precision
(Analyst-II)
HPLC Instrument No. GPR/AD/I-051 GPR/AD/I-021
Date of analysis 02.11.2016 03.11.2016
HPLC column No. C18-525 C18-474
Sample No. % Assay
1 97.2 97.0
2 97.2 97.0
3 97.1 97.0
4 97.0 97.0
5 97.2 97.0
6 97.1 97.0

54. 54
ROBUSTNESS:
Parameters Values
Retention
Time
Tailing factor
Theoretical
plates
% RSD of
standard
area
%Assay
Abs
olut
e
diffe
renc
e
Control
As per
method
3.74 1.2 3481 0.12 97.0 NA
Flow rate
(± 0.1 mL/min)
0.9 mL/min 4.08 1.2 3626 0.04 96.9 0.1
1.1mL/min 3.39 1.2 3131 0.10 97.0 0.0
Change in
Wavelength(± 2
nm)
288 nm 3.74 1.2 3481 0.13 97.0 0.0
292 nm 3.74 1.2 3481 0.13 97.0 0.0
Buffer pH(±0.2
unit)
pH-3.8 3.92 2.0 6694 0.09 97.6 0.9
pH-4.2 3.88 1.9 6384 0.07 97.6 0.7

55. 55
SUMMARY AND DISCUSSION:
RP-HPLC method has shown adequate separation for Midodrine Hydrochloride
from its degradation product. Separation has achieved on a Zorbax Eclipse C18
(150×4.6 mm), 3.5µm column at 25°C temperature by using buffer pH 6.8:ACN
(95:5) as mobile phase at a flow rate of 1.0 ml/min, and UV detection at 290nm. In
the present study, comprehensive stress testing of Midodrine Hydrochloride was
carried out according to ICH guideline Q1A (R2).The specificity of the method was
determined by assessing interference from the placebo and by stress testing of the
drug (forced degradation). There was no other eluting, interfering peak from
excipients, impurities or degradation product due to variable stress conditions and
the method was found to be specific for estimation of Midodrine Hydrochloride . The
method was validated in term of specificity, linearity, accuracy, precision, and
robustness. The linearity of the proposed method was found to be r2 = 1.000 for
Atazanavir sulfate. Accuracy determined by recovery study found 101.6.

56. 56
CONCLUSION:
The method is specific for the determination of % Assay of Midodrine
Hydrochloride in Midodrine Hydrochloride Oral solution, 0.5mg/mL.
the method is precise and accurate across the suitable analytical range.
Standard solution has been seen to be stable for 75 hours at benchtop
condition and 73 hours at 10°C. The sample solution has been seen to be
stable for 73 hours at benchtop condition and 72 hours at 10°C.
the method is robust towards deliberate minor changes in the method
parameters of both HPLC and Assay.
The method can be used in quality control laboratory for release of
production batches and stability study.
The developed stability-indicating assay method was found to be simple,
accurate, sensitive, precise, specific, and rapid. This method can be applied
for routine quantitative analysis of Midodrine Hydrochloride in bulk and
pharmaceutical formulation like an oral solution. This method was also used
to check the quality of the product after different storage conditions and when
stress degradation is carried out.

57. REFERENCES
1. http://en.wikipedia.org wiki//
2. www.drugbank.com//
3. https://dailymed.nlm.nih.gov/
4. www.google.co.in/hplc
5. Hemant K. Jain*, Vishal K. Nikam Development And Validation Of
HPTLC Method For Determination Apixaban In Bulk And Tablets, Int J
App Pharm, Vol 9, Issue 5, 2017, 78-82.
6. Hemant K. Jain*, Vishal K. Nikam Formulation Development And Stability
Indicating HPLC Assay Of Tablets Of Apixaban, Int J Pharm Sci, Vol 9,
Issue 10, 2017,24-32
7. Hemant K. Jain* Kishor N. Gujar And Varsha A. Randhe first order
derivative spectrophotometric method development and validation for
Midodrine hydrochloride in bulk and tablets World Journal of Pharmacy
and Pharmaceutical Sciences ,2016, volume 5, issue 7, 1760-1767.

58. CONT..
8. Hemant K. Jain* stability indicating Rp-Hplc assay method for estimation
of Midodrine hydrochloride in bulk and tablets Int J Pharm Sci,2016, Vol
8, Issue 9, 283-287.
9. Dr. Mrinalini C. Damle* Sunny R. Salunke Stability-Indicating HPTLC
Method For Determination Of Midodrine Hydrochloride European
Journal Of Pharmaceutical And Medical Research, 2016,3(9), 202-207.
10. Pritam S. Jain* Prashant V. Bhadane, Harshal P. Chaudhari, Sanjay J
Surana,
Area under Curve Method Development and Validation of Midodrine
Hydrocholride, International Journal of Pharmaceutical Chemistry and
Analysis; October-December 2015;2(4):154-160.
11. Mamdouh R.Rezk* Osama Abdel Sattar, Amr M.Badawy, Omaima
M.Khattab, selective determination of Midodrine hydrochloride in the
presence of its Acidic Degradation Product Analytical Chemistry An
Indian Journal , volume ,12 issue, 2013 [182-187].

59. 59