A PRESENTATION ON :
DEVELOPMENT AND VALIDATION OF AN RP-HPLC
METHOD FOR SIMULTANEOUS DETERMINATION OF
RAMIPRIL AND AMLODIP...
INTRODUCTION:
RAMIPRIL: ACE inhibitor, anti hypertensive
2
ANGIOTENSINOGEN
ANGIOTENSIN-1
ANGIOTENSIN-2
AT1 RECEPTOR
ALDOST...
AMLODIPINE: It is the drug that blocks L-type of voltage gated
calcium channel present in blood vessels and heart
3
WHY TO GIVE DRUG IN COMBINATION…..?
4
 Large majority of hypertensives ultimately require drug
combination to decrease the damage of heart, brain, kidney,
etc....
DRUG PROFILE
6
RAMIPRIL AMLODIPINE
STUCTURE
CHEMICAL
NAME
[(2S, 3aS, 6aS)-1-[(S)-2-
[[(S)-1-
(ethoxycarbonyl)-3-
phenylpro...
7
Chemical structure of impurities:
Ramipril impurities A (II), B (III), C (IV), D
(V)
Amlodipine impurity D
OBJECTIVE OF WORK:
 To develop HPLC method for determination of Ramipril and
Amlodipine in the same tablet dosage form.
...
PLAN OF WORK:
9
Literature survey
Selection and procurement of drug
study of physical properties of drug
Optimization of a...
REAGENTS AND CHEMICALS
SL
NO
NAME OF THE CHEMICALS MAKE
1 RP API Green syn co., ltd
2 AL besylate API weihai disu pharmc.C...
HPLC INSTRUMENTS AND ANALYTICAL
CONDITION:
 Analysis was performed on a chromatographic system of
shimadzu prominence con...
 Cont’d….
 Data acquisition was made with shimadzu LC solution
software.
 Mobile phase A consisted of 60mM sodium perch...
Sample preparation:
13
Twenty tablets are powdered
25 mg of RP and 50 mg of AL was weighed and dissolved in mobile phase A...
 Samples were subjected to stressed conditions of light, heat,
acid, base and oxidation.
 In the stress condition, all t...
METHOD DEVELOPMENT
15
Gradient HPLC method was adopted
 To get a shorter run time
 Higher sensitivity.
 Stainless steel...
 As RP and AL had N-H groups as basic nitrogen centers,
the amount of perchlorate and the pH of M.P would affect
the peak...
17
Gradient program proposed for the analysis of AL (Amlodipine), RP
(Ramipril) and their related substances
18
Chromatograms of sample solution (1mg/mL RP and 2mg/mL AL solution using
the final optimized mehtod).1.Benzene sulfonic...
METHOD VALIDATION:
VALIDAT
ED WITH
SYSTEM
SUITABILIT
Y
SPECIFICTY
LINEARITY
RANGE
ACCURACYPRECISION
LOD AND LOQ
STABILITY
...
System suitability test:
 The solution containing 0.1mg/mL of RP, 0.2mg/mL of AL
besylate and each 0.1 mg/mL of five majo...
Acceptance criteria:
21
Less than 2% RSD
for peak area
Greater than 3000
column plates
Less than 1.5 of the
USP tailing fa...
22
Chromatogram of the system suitability test solution. 1. Benzene sulfonic acid, 2.
Ramipril impurity A, 3. Amlodipine i...
Specificity :
 The selectivity of the method was confirmed by observing
potential interference caused by excipients of ta...
Linearity and range:
 A standard stock solution containing 0.25mg/mL of RP and
0.5mg/mL of AL besylate was prepared by mo...
 The linearity was checked by analyzing six working
solution of RP over the concentration range 0.01 – 0.25
mg/mL and 0.0...
Accuracy and precision:
 Known amount of each standard powder was added to
blank sample
 Mixed , extracted and subsequen...
27
Accuracy results [Recovery (%)] for the determination of AL
(Amlodipine) and RP (Ramipril).
 Repeatability or intra – day precision was investigated by
injecting six replicate sample solution on the same day.
 In...
LOQ and LOD:
 The LOQs for RP and AL corresponding to a signal-to-noise
ratio of 10 were 0.2µg/mL and 0.07 µg/mL.
 The L...
Stability of solutions and robustness:
 The stability of the standard stock solutions was determined by
quantitatively de...
Robustness :
 The robustness of the method was investigated by a small
variety of conditions including changes of pH of t...
32
Chromatographic parameter setting applied in the
robustness investigation
FORCED DEGRADATION STUDIES
Degradation profile under different stress condition:
1. Acid Degradation (HCL)
2. Alkali Degra...
34
 Acid Degradation: It was conducted using 2mL of 0.1M
hydrochloric acid
 Alkali Degradation: It was carried out in 2mL of 0.1M
sodium hydroxide.
35
 Oxidation Degradation: It was performed by adding 2mL of
3% H2O2 and kept in water bath for 5min, and then diluted
with ...
 Thermal Degradation: For the temperature stress study,
compound RPAL tablets, RP and AL besylate API were exposed
to dry...
 Photo Degradation : For photo stability studies composed
RPAL tablets , RP and AL besylate API were exposed t 4500
lx li...
 All stressed samples were compare with an un-stressed
sample solution.
 The peak purity indices for RP and AL in stress...
 Origination of related substance:
 The origination of the related substances in tablets was
investigated.
 The peaks n...
41
Chromatograms of RP (Ramipril) API and AL (Amlodipine) besylate API .
Assay of AL and RP in tablets:
 Three batches of compound tablets were analysed using
the developed method.
42
Conclusion :
 A gradient LC method has been developed and validated for
the analysis of RP and AL in tablet dosage forms....
 Therefore, the chromatographic method can be used to
analyze samples obtained during accelerated stability
experiments a...
45
Upcoming SlideShare
Loading in …5
×

DEVELOPMENT AND VALIDATION OF AN RP-HPLC METHOD FOR SIMULTANEOUS DETERMINATION OF RAMIPRIL AND AMLODIPINE IN TABLETS

6,526 views

Published on

DEVELOPMENT AND METHOD VALIDATION

Published in: Science
2 Comments
9 Likes
Statistics
Notes
No Downloads
Views
Total views
6,526
On SlideShare
0
From Embeds
0
Number of Embeds
15
Actions
Shares
0
Downloads
426
Comments
2
Likes
9
Embeds 0
No embeds

No notes for slide

DEVELOPMENT AND VALIDATION OF AN RP-HPLC METHOD FOR SIMULTANEOUS DETERMINATION OF RAMIPRIL AND AMLODIPINE IN TABLETS

  1. 1. A PRESENTATION ON : DEVELOPMENT AND VALIDATION OF AN RP-HPLC METHOD FOR SIMULTANEOUS DETERMINATION OF RAMIPRIL AND AMLODIPINE IN TABLETS 1 PRESENTED BY: AMPATI RAHUL {M PHARMACY:PHARMACEUTICAL ANALYSIS}
  2. 2. INTRODUCTION: RAMIPRIL: ACE inhibitor, anti hypertensive 2 ANGIOTENSINOGEN ANGIOTENSIN-1 ANGIOTENSIN-2 AT1 RECEPTOR ALDOSTERONE SALT AND WATER RETENTION INCREASES BP RENIN ACE RAMIPR IL - - - - -
  3. 3. AMLODIPINE: It is the drug that blocks L-type of voltage gated calcium channel present in blood vessels and heart 3
  4. 4. WHY TO GIVE DRUG IN COMBINATION…..? 4
  5. 5.  Large majority of hypertensives ultimately require drug combination to decrease the damage of heart, brain, kidney, etc.,  Fixed dose combinations of drugs with complementary properties offer the advantages of simplicity, tolerability, convenience and cost effectiveness, as well as the compliance.  The combination therapy of ACEI and CCB has proved to be effective.  So the combination of RP and AL would also be good therapeutic option. 5
  6. 6. DRUG PROFILE 6 RAMIPRIL AMLODIPINE STUCTURE CHEMICAL NAME [(2S, 3aS, 6aS)-1-[(S)-2- [[(S)-1- (ethoxycarbonyl)-3- phenylpropyl] amino] propanoyl] octahydro cyclopenta [b] pyrrole-2- carboxylic acid [3-Ethyl5-methyl(4RS)-2-[(2- aminoethoxy) methyl]-4-(2-chlorophenyl)- 6-methyl-1,4- dihydropyridine-3,5- dicarboxylate SIDE EFFECTS Hypotension, cough Peripheral edema
  7. 7. 7 Chemical structure of impurities: Ramipril impurities A (II), B (III), C (IV), D (V) Amlodipine impurity D
  8. 8. OBJECTIVE OF WORK:  To develop HPLC method for determination of Ramipril and Amlodipine in the same tablet dosage form.  To validate the developed methods as per the ICH guidelines.  To study stability indicating method of drug. 8
  9. 9. PLAN OF WORK: 9 Literature survey Selection and procurement of drug study of physical properties of drug Optimization of analytical method validation of developmental method Forced degradation studies Result and discussion Summary and conclusion
  10. 10. REAGENTS AND CHEMICALS SL NO NAME OF THE CHEMICALS MAKE 1 RP API Green syn co., ltd 2 AL besylate API weihai disu pharmc.Co.,ltd 3 Tablet exepients Chengdu haisco pharmaceutical co.,ltd 4 RS of AL beyslate National institute for control of pharmaceutical products 5 RS of RP European directorate 6 Impurity standards of RP and AL European directorate 7 HPLC-grade acetonitrile Honey well 8 HPLC-grade triethylamine Kermel chemical reagents company 10
  11. 11. HPLC INSTRUMENTS AND ANALYTICAL CONDITION:  Analysis was performed on a chromatographic system of shimadzu prominence consisting of AT liquid pump and PDA detector with manual 20 µl sample injection loop.  Chromatographic seperation was achieved on Inertsil ODS -3 (250mm * 4.0mm , 3µm) column.  The column temperature was maintained at 55°C and flow rate was 1.0mL/min.  The detection wavelength was set at 210nm. 11
  12. 12.  Cont’d….  Data acquisition was made with shimadzu LC solution software.  Mobile phase A consisted of 60mM sodium perchlorate buffer (containing 7.2mM triethylamine)- acetonitrile(60:40,v/v)  Mobile phase B was 60mM sodium perchlorate buffer (containing 7.2mM triethylamine)- acetonitrile (20:80,v/v).  The apparent pH of the mobile phase was adjusted to 2.6 with phosphoric acid. 12
  13. 13. Sample preparation: 13 Twenty tablets are powdered 25 mg of RP and 50 mg of AL was weighed and dissolved in mobile phase A Ultra sonicated for 15mins The mixture was centrifuged at 10000 rpm for 10mins. The supernatant was separated and transferred into HPLC instrument to be analyzed
  14. 14.  Samples were subjected to stressed conditions of light, heat, acid, base and oxidation.  In the stress condition, all the solutions were prepared by weighing 4-tablets equivalent mass of sample powders containing about 10mg RP or 20mg AL pure API. 14
  15. 15. METHOD DEVELOPMENT 15 Gradient HPLC method was adopted  To get a shorter run time  Higher sensitivity.  Stainless steel column(250mm*4.0mm, 3µm) was used (ph. Eur.).  Mobile phase A and B all consisted of acetonitrile, Sodium perchlorate buffer and 1mL trimethylamine with different pH values adjusted to 3.6 and 2.6 with phosphoric acid.
  16. 16.  As RP and AL had N-H groups as basic nitrogen centers, the amount of perchlorate and the pH of M.P would affect the peak shape, resolution and symmetry.  The results shows that the retention time of RP and AL were lengthened when the amount of perchlorate increased.  The RT of RP would be shortened while the RT of AL has no significant change when the pH was decreased.  Column diameter of 3µm was chosen  Column temperature of 60°C, 55°C and 50°C were studied and 55°C was finally chosen. 16
  17. 17. 17 Gradient program proposed for the analysis of AL (Amlodipine), RP (Ramipril) and their related substances
  18. 18. 18 Chromatograms of sample solution (1mg/mL RP and 2mg/mL AL solution using the final optimized mehtod).1.Benzene sulfonic acid, 2. Unknown 1, 3.Unknown 2, 4.Amlodipine impurity D, 5.Unknown 3, 6.Ramipril impurity B, 7.Unknown 4, 8.Ramipril impurity C, 9. Unknown 5, 10.Unknown 6, 11.Ramipril impurity D, 12.Unknown 7, and 13.Unknown 8.
  19. 19. METHOD VALIDATION: VALIDAT ED WITH SYSTEM SUITABILIT Y SPECIFICTY LINEARITY RANGE ACCURACYPRECISION LOD AND LOQ STABILITY ROBUSTNESS 19
  20. 20. System suitability test:  The solution containing 0.1mg/mL of RP, 0.2mg/mL of AL besylate and each 0.1 mg/mL of five major impurities was prepared by mobile phase A.  System suitability was determined by six replicate injections of the system suitability solution.  The resolutions among RP, AL and the closest eluting peaks were bigger than 2 which indicating that this method was reliable for the quantitation of RP and AL 20
  21. 21. Acceptance criteria: 21 Less than 2% RSD for peak area Greater than 3000 column plates Less than 1.5 of the USP tailing factor Greater than 1.5 of the resolution
  22. 22. 22 Chromatogram of the system suitability test solution. 1. Benzene sulfonic acid, 2. Ramipril impurity A, 3. Amlodipine impurity D, 4. Ramipril impurity B, 5. Ramipril impurity C, and 6. Ramipril impurity D
  23. 23. Specificity :  The selectivity of the method was confirmed by observing potential interference caused by excipients of tablet formulations and degradation products .  The chromatogram of the tablet excipients shows that there were no interference of peaks to the determination of Rp and AL. 23
  24. 24. Linearity and range:  A standard stock solution containing 0.25mg/mL of RP and 0.5mg/mL of AL besylate was prepared by mobile phase A Dilute with the same solvent to yield solution at different concentrations These solutions were protected from light using aluminium foil Stored at 4ºC in the refrigerator 24
  25. 25.  The linearity was checked by analyzing six working solution of RP over the concentration range 0.01 – 0.25 mg/mL and 0.014 – 0.36 mg/mL for AL  Results : 25 RP AL y=44164x + 303.8 y= 37963x - 597 r2= 09998 r2= 0.9997
  26. 26. Accuracy and precision:  Known amount of each standard powder was added to blank sample  Mixed , extracted and subsequently dilute to yield three different concentrations of each drug  RP: 0.08, 0.10 and 0.12 mg/mL  AL: 0.16, 0.20 and 0.24 mg/mL 26
  27. 27. 27 Accuracy results [Recovery (%)] for the determination of AL (Amlodipine) and RP (Ramipril).
  28. 28.  Repeatability or intra – day precision was investigated by injecting six replicate sample solution on the same day.  Inter day precision was assessed by analysis newly prepare sample solutions in triplicate over three consecutive days. 28 RP AL RSD% RSD% Intra day 0.46 0.55 Inter day 1.60 1.80
  29. 29. LOQ and LOD:  The LOQs for RP and AL corresponding to a signal-to-noise ratio of 10 were 0.2µg/mL and 0.07 µg/mL.  The LODs corresponding to a signal to noise ratio of 3 were 0.06µg/mL and 0.2 µg/mL.  The resultant RSD values for these studies were < 3.5% 29
  30. 30. Stability of solutions and robustness:  The stability of the standard stock solutions was determined by quantitatively determining each drug in different time comparing to the response obtained for freshly prepared standard solutions.  The stability of sample solution was tested for every 2h interval upto 12h.  The RSD values of RP and AL were 1.2% and 1.0%, respectively. 30
  31. 31. Robustness :  The robustness of the method was investigated by a small variety of conditions including changes of pH of the eluent, flow rate and column temperature .  The assay results for RP and AL by three different analysts in the same laboratory were also investigated.  The RSD values did not exceed 2.5%.  The degree of reproducibility of the results obtained as a result of small deliberate variations in the method parameters and by changing analytical operators demonstrated that the method was robust. 31
  32. 32. 32 Chromatographic parameter setting applied in the robustness investigation
  33. 33. FORCED DEGRADATION STUDIES Degradation profile under different stress condition: 1. Acid Degradation (HCL) 2. Alkali Degradation (NaOH) 3. Oxidation Degradation (H2O2) 4. Thermal Degradation 5. Photo Degradation 33
  34. 34. 34  Acid Degradation: It was conducted using 2mL of 0.1M hydrochloric acid
  35. 35.  Alkali Degradation: It was carried out in 2mL of 0.1M sodium hydroxide. 35
  36. 36.  Oxidation Degradation: It was performed by adding 2mL of 3% H2O2 and kept in water bath for 5min, and then diluted with mobile phase A. 36
  37. 37.  Thermal Degradation: For the temperature stress study, compound RPAL tablets, RP and AL besylate API were exposed to dry heat of 60°C in a convention oven for 10 days. 37
  38. 38.  Photo Degradation : For photo stability studies composed RPAL tablets , RP and AL besylate API were exposed t 4500 lx light in a light cabinet for 10days. 38
  39. 39.  All stressed samples were compare with an un-stressed sample solution.  The peak purity indices for RP and AL in stressed solutions were found to be better ( purity angle < purity threshold)  And they also evidence the ability of the method to access unequivocally the analytes of interest in the presence of potential interference. 39
  40. 40.  Origination of related substance:  The origination of the related substances in tablets was investigated.  The peaks numbered 6, 8, 11 as defined in the chromatograms of sample solution( RL impurity B, C, D respectively, according to the RRT) would same from RP API.  In addition to the known impurities, the unknown impurities peaks numbered 2, 9, 10, 12, 13 originated in RP API  And the peaks numbered 1 ( benzoic acid), 3, 4(Amlodipine impurity D), 5 and 7 would originate in AL besylate API. 40
  41. 41. 41 Chromatograms of RP (Ramipril) API and AL (Amlodipine) besylate API .
  42. 42. Assay of AL and RP in tablets:  Three batches of compound tablets were analysed using the developed method. 42
  43. 43. Conclusion :  A gradient LC method has been developed and validated for the analysis of RP and AL in tablet dosage forms.  The results of the stress testing revealed that the method was specific and selective.  The proposed method has the ability to separate the two main components from their degradation products, related substances found in tablet dosage forms and the tablet excipients. 43
  44. 44.  Therefore, the chromatographic method can be used to analyze samples obtained during accelerated stability experiments and routine assay of RP and AL in combined tablet dosage forms.  In addition, the procedure can be further applied for the detection and determination of the related substances in tablets 44
  45. 45. 45

×